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The development of a second malignancy after the diagnosis of childhood acute lymphoblastic leukemia (ALL) is a rare event. Certain second malignancies have been linked with specific elements of leukemia therapy, yet the etiology of most second neoplasms remains obscure and their optimal management strategies are unclear. This is a first comprehensive report of non-Hodgkin lymphomas (NHLs) following pediatric ALL therapy, excluding stem-cell transplantation. We analyzed data of patients who developed NHL following ALL diagnosis and were enrolled in 12 collaborative pediatric ALL trials between 1980-2018. Eighty-five patients developed NHL, with mature B-cell lymphoproliferations as the dominant subtype (56 of 85 cases). Forty-six of these 56 cases (82%) occurred during or within 6 months of maintenance therapy. The majority exhibited histopathological characteristics associated with immunodeficiency (65%), predominantly evidence of Epstein-Barr virus-driven lymphoproliferation. We investigated 66 cases of post-ALL immunodeficiency-associated lymphoid neoplasms, 52 from our study and 14 additional cases from a literature search. With a median follow-up of 4.9 years, the 5-year overall survival for the 66 patients with immunodeficiency-associated lymphoid neoplasms was 67.4% (95% confidence interval [CI], 56-81). Five-year cumulative risks of lymphoid neoplasm- and leukemia-related mortality were 20% (95% CI, 10.2-30) and 12.4% (95% CI, 2.7-22), respectively. Concurrent hemophagocytic lymphohistiocytosis was associated with increased mortality (hazard ratio, 7.32; 95% CI, 1.62-32.98; P = .01). A large proportion of post-ALL lymphoid neoplasms are associated with an immunodeficient state, likely precipitated by ALL maintenance therapy. Awareness of this underrecognized entity and pertinent diagnostic tests are crucial for early diagnosis and optimal therapy., (© 2023 by The American Society of Hematology.)

Δ6-Methyltestosterone was reported as the main active ingredient of the purported "dietary supplement" Jungle Warfare. This compound is structurally similar to 17α-methyltestosterone, containing an additional Δ6 double bond, and is reported to possess notable androgenic activity, raising concerns over the potential for abuse of Jungle Warfare in sport. The in vivo metabolism of Δ6-methyltestosterone in greyhounds was investigated. Urinary phase I (unconjugated) and phase II (glucuronide) metabolites were detected following oral administration using liquid chromatography-mass spectrometry. No phase II sulfate metabolites were detected. The major phase I metabolite was confirmed as 16α,17β-dihydroxy-17α-methylandrosta-4,6-dien-3-one by comparison with a synthetically-derived reference material. Minor amounts of the parent drug were also confirmed. Glucuronide conjugated metabolites were also observed, but were found to be resistant to hydrolysis using the Escherichia coli β-glucuronidase enzyme. Qualitative excretion profiles, limits of detection, and extraction recoveries were determined for the parent drug and the major phase I metabolite. These results provide a method for the detection of Jungle Warfare abuse in greyhounds suitable for incorporation into routine screening methods conducted by anti-doping laboratories., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Context: Integrative medicine (IM) is a rapidly growing field whose providers report clinical success in treating significant stress, chronic pain, and depressive and anxiety symptoms. While IM therapies have demonstrated efficacy for numerous medical conditions, IM for psychological symptoms has been slower to gain recognition in the medical community., Objective and Design: This large, cross-sectional study is the first of its kind to document the psychosocial profiles of 4182 patients at 9 IM clinics that form the BraveNet Practice-Based Research Network (PBRN)., Results: IM patients reported higher levels of perceived stress, pain, and depressive symptoms, and lower levels of quality of life compared with national norms. Per provider reports, 60% of patients had at least one of the following: stress (9.3%), fatigue (10.2%), anxiety (7.7%), depression (7.2%), and/or sleep disorders (4.8%). Pain, having both physiological and psychological components, was also included and is the most common condition treated at IM clinics. Those with high stress, psychological conditions, and pain were most frequently treated with acupuncture, IM physician consultation, exercise, chiropractic services, diet/nutrition counseling, and massage., Conclusion: With baseline information on clinical presentation and service utilization, future PBRN studies can examine promising interventions delivered at the clinic to treat stress and psychological conditions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The objective of the Biomarkers of Nutrition for Development (BOND) project is to provide state-of-the-art information and service with regard to selection, use, and interpretation of biomarkers of nutrient exposure, status, function, and effect. Specifically, the BOND project seeks to develop consensus on accurate assessment methodologies that are applicable to researchers (laboratory/clinical/surveillance), clinicians, programmers, and policy makers (data consumers). The BOND project is also intended to develop targeted research agendas to support the discovery and development of biomarkers through improved understanding of nutrient biology within relevant biologic systems. In phase I of the BOND project, 6 nutrients (iodine, vitamin A, iron, zinc, folate, and vitamin B-12) were selected for their high public health importance because they typify the challenges faced by users in the selection, use, and interpretation of biomarkers. For each nutrient, an expert panel was constituted and charged with the development of a comprehensive review covering the respective nutrient's biology, existing biomarkers, and specific issues of use with particular reference to the needs of the individual user groups. In addition to the publication of these reviews, materials from each will be extracted to support the BOND interactive Web site (http://www.nichd.nih.gov/global_nutrition/programs/bond/pages/index.aspx). This review represents the first in the series of reviews and covers all relevant aspects of iodine biology and biomarkers. The article is organized to provide the reader with a full appreciation of iodine's background history as a public health issue, its biology, and an overview of available biomarkers and specific considerations for the use and interpretation of iodine biomarkers across a range of clinical and population-based uses. The review also includes a detailed research agenda to address priority gaps in our understanding of iodine biology and assessment., (© 2014 American Society for Nutrition.)

Objective: Doctors find patients with medically unexplained symptoms (MUS) challenging to manage and some hold negative attitudes towards these patients. It is unknown when and how these views form. This study examines medical trainees' beliefs and influences about MUS., Methods: Semi-structured interviews with 43 medical trainees. Using an iterative approach, initial emergent themes were explored in subsequent interviews. Data generation continued until thematic saturation was achieved., Results: Participants had received no training in MUS but had developed views about causes and management. They struggled with the concept of 'diagnosis by exclusion'. Attitudes towards patients had developed through informal clinical observation and interactions with doctors. Many welcomed formal training but identified a need to integrate theoretical learning with clinical application., Conclusion: Despite limited teaching, medical trainees are aware of the challenges in diagnosing and managing patients with MUS, acquiring attitudes through a hidden curriculum. To be welcomed, training must be evidence-based, theoretically informed, but clinically applicable., Practical Implications: Current medical training fails to equip doctors to engage with MUS and potentially fosters the development of unhelpful views of these patients. Informed teaching on diagnosis and management of MUS is necessary at a trainee level to limit the development of negative attitudes., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Purpose: To examine associations between biomarkers of joint tissue metabolism and whole blood lead (Pb), separately for men and women in an African American and Caucasian population, which may reflect an underlying pathology., Methods: Participants in the Johnston County Osteoarthritis Project Metals Exposure Sub-Study (329 men and 342 women) underwent assessment of whole blood Pb and biochemical biomarkers of joint tissue metabolism. Urinary cross-linked N telopeptide of type I collagen (uNTX-I) and C-telopeptide fragments of type II collagen (uCTX-II), serum cleavage neoepitope of type II collagen (C2C), serum type II procollagen synthesis C-propeptide (CPII), and serum hyaluronic acid (HA) were measured using commercially available kits; the ratio of [C2C:CPII] was calculated. Serum cartilage oligomeric matrix protein (COMP) was measured by an in-house assay. Multiple linear regression models were used to examine associations between continuous blood Pb and biomarker outcomes, adjusted for age, race, current smoking status, and body mass index. Results are reported as estimated change in biomarker level for a 5-unit change in Pb level., Results: The median Pb level among men and women was 2.2 and 1.9μg/dL, respectively. Correlations were noted between Pb levels and the biomarkers uNTX-I, uCTX-II, and COMP in women, and between Pb and uCTX-II, COMP, CPII, and the ratio [C2C:CPII] in men. In adjusted models among women, a 5-unit increase in blood Pb level was associated with a 28% increase in uCTX-II and a 45% increase in uNTX-I levels (uCTX-II: 1.28 [95% CI: 1.04-1.58], uNTX-I: 1.45 [95% CI:1.21-1.74]). Among men, levels of Pb and COMP showed a borderline positive association (8% increase in COMP for a 5-unit change in Pb: 1.08 [95% CI: 1.00-1.18]); no other associations were significant after adjustment., Conclusions: Based upon known biomarker origins, the novel associations between blood Pb and biomarkers appear to be primarily reflective of relationships to bone and calcified cartilage turnover among women and cartilage metabolism among men, suggesting a potential gender-specific effect of Pb on joint tissue metabolism that may be relevant to osteoarthritis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The interactions of tryptophan and its peptide homologues with thiophilic ligands were studied in terms of their chromatographic retention and steady-state fluorescence under various conditions, and compared with non-polar structures typically regarded as pure hydrophobic ligands. The experimental results show that both non-polar and polar interactions are involved in what has been termed "thiophilic adsorption chromatography".

Colloidal polymer particles are widely used in a variety of applications ranging from chromatography to surface modified bioreactors in protein arrays. In the present study, surface attachment of polystyrene particles to a polystyrene substrate has been performed using oligonucleotide hybridization. Thiolated complementary oligomers of cytosine and guanine have been covalently coupled to a pyridyl disulphide (PDS) modified polyethyleneglycol tether, forming part of a triblock copolymer which is adsorbed to the polystyrene surfaces via hydrophobic polypropylene oxide center blocks. The ability to withstand shear forces was studied using a laminar flow cell and the uptake of oligomers on the particles was quantified using two complementary techniques: UV-spectroscopy and sedimentation field flow fractionation. The possibility to tether particles in a flow cell suitable for practical use in e.g. a FIA-system is demonstrated.

In exploratory scale biotechnological process development, the product must be rapidly identified although a reference compound may not always be available. LC-diode-array detection and MS were used for this purpose in a process producing 9alpha-hydroxyprogesterone from progesterone as substrate. The electrospray ionization mass spectrometer was combined with an ion trap mass spectrometer for the second generation MS. The preliminary identification, which could be carried out within the course of a day, confirmed that the product was a hydroxyprogesterone. The final identification step, which was much more material intensive and hence time consuming, involved a two-step preparative separation to yield quantities necessary for definitive product identification based on 1H- and 13C NMR.

We investigated a novel strategy for measuring the synthesis rate of proteins in skeletal and cardiac muscle. Mass isotopomer distribution analysis allows measurement of the isotopic enrichment of the true biosynthetic precursor for proteins (tRNA-amino acids), but cannot easily be applied to slow turnover muscle proteins due to insufficient isotope incorporation into multiply labeled species. Using a rapid turnover protein from the same tissue, however, might reveal tRNA-amino acid enrichment. We tested this strategy in rats on muscle creatine kinase (CK). A trypsinization peptide (3647u) containing 5 leucine repeats was identified by computer-simulated digestion of CK and then isolated from trypsin hydrolysates. Mass isotopomer abundances were determined by electrospray ionization-magnetic sector-mass spectrometry after in vivo administration of [(2)H(3)]leucine. Myosin heavy chain was also isolated and hydrolyzed to free amino acids. Muscle tRNA-amino acids were well labeled, by direct measurement. Enrichments of M(+1) and M(+2) mass isotopomers in the CK-peptide were measurable but low (consistent with a CK half-life of 3-10 days). Incorporation into skeletal muscle myosin indicated a half-life of 54 days. In conclusion, the general strategy of measuring protein kinetics by quantifying mass isotopomer abundances of mid-sized peptides from protein hydrolysates is effective, but CK does not turn over rapidly in muscle, contrary to previous reports. Identification of a rapid turnover muscle protein would be useful for this purpose.

The effects of carrier ionic strength and electrolyte composition on the retention of poly(ethylene oxide) in aqueous flow field-flow fractionation have been investigated in this work. The study shows retention to be particularly sensitive to the presence of salts, as well as to the nature of the cation. Specifically, retention effects due to sample load are found to be very different in solutions containing potassium salts compared to those observed in solutions of the corresponding sodium salts. In a potassium-containing medium, the dependence of retention on sample mass is similar to that found previously for polyelectrolytes. This effect, which is particularly prominent for samples of low molecular mass, can be attributed to specific interactions between cation and polymer.

The measurement of protein kinetics by isotopic techniques has been hindered by the long-standing difficulty of accurately measuring the isotope content of the biosynthetic precursor pool (aminoacyl-tRNA in tissues). Mass isotopomer distribution analysis (MIDA) is a stable isotope-mass spectrometric (MS) technique for measuring biosynthetic precursor enrichments from measurements on a polymeric product, based on combinatorial probabilities of labeled and unlabeled monomeric subunits. Proteins contain complex isotopomer patterns as a result of their relatively high molecular mass, however, so that resolution of the individual mass isotopomers in the polymeric product (an analytic requirement for MIDA) is technically difficult. An approach for measuring protein synthesis by MIDA is described and tested here: First, in vitro, using a synthetic peptide present in human serum albumin; and then, in vivo, for albumin synthetic rate in rats. A peptide contained in human serum albumin (SVVLLLR) and theoretically recoverable from trypsin/chymotrypsin proteolysis was synthesized by solid-phase peptide synthesis using known mixtures of natural abundance and [5,5, 5-2H3]leucine. Additionally, enriched and natural abundance peptides were mixed in vitro to simulate in vivo biosynthesis and to address problems of instrument accuracy, precision, and data management. The mass isotopomer patterns of the synthetic peptides were analyzed using electrospray ionization (ESI) with both magnetic sector and quadrupole mass analyzers. The resolution of the magnetic sector was superior to that of the quadrupole instrument, but accuracy and precision in the measurement of mass isotopomer abundances and kinetic parameters were comparable and both gave values close to those predicted. Next, rats were infused with [5,5,5-2H3]leucine intravenously, and a leucine-rich peptide was isolated and purified from trypsin-digested rat serum albumin (RHPDYSVSLLLR, 1456 Da) and then analyzed by ESI-MS using a magnetic sector instrument. Precursor pool enrichments and fractional synthetic rates (0.45 +/- 0.03 day-1, t1/2 = 1.53 +/- 0.09 days) were calculated. Biosynthetic rates of rat serum albumin were congruent with previously published values. In summary, measurement of protein synthesis and precursor pool enrichments by MIDA is technically feasible and practical in vivo using proteolytically derived peptides and ESI-MS analysis., (Copyright 1999 Academic Press.)

Steric/hyperlayer field-flow fractionation (FFF) is an established analytical technique for separating and characterizing particles in the 1-100 microns diameter range. The separation can be based on differences in size, density, shape and mechanical properties of the particles. In the course of an analysis of the water transporter system of Chinese hamster ovary (CHO) cells and one of their high permeability mutants, the first successful attempt was made to use the steric/hyperlayer FFF system for the purpose of separating particles based on a time-dependent property, namely, the differential swelling of the two cell types. The present study was undertaken to simulate numerically the separation and suggest selection of operating conditions to minimize repetitive experiments. The computer simulation was developed using Maple V, a symbolic computing environment. It is shown that the model is able to predict an optimal velocity of carrier buffer that maximizes resolution. Predicted velocity/resolution pairs are in good agreement with available experimental data. Empirical models for the lift forces encountered in such FFF experiments, and for the zone broadening observed in work with cell sized particles, form the basis for this model.

Thin-layer chromatography (TLC) is one of the simplest and most convenient techniques to separate small molecules. Of a variety of TLC separation modes, only size-exclusion was successfully used to separate proteins. In this paper, adsorption-TLC was used to separate proteins. The net charges were calculated for four model proteins, albumin, transferrin, lactoferrin and lysozyme, under different pH values. The suitable pH values for separation were determined according to the results from such calculations. Then, the adsorption isotherms of the four proteins were measured to deduce the ionic strength for appropriate elution conditions. Optimal conditions, 0.01 M bicine and pH 8.50, and a three-step elution process (1st step 0.01 M NaCl, 2nd 0.025 M NaCl, and 3rd 0.10 M NaCl), were obtained. Finally, the four model proteins were successfully separated under these elution condition.

This study examines the size and compositional heterogeneity of particles in a commercial lipid emulsion (Intralipid) before and after equilibration with penclomedine, a highly lipophilic cytotoxic agent. Emulsions were fractionated by sedimentation field-flow fractionation (sedFFF), and particle sizes of the monodisperse fractions were determined by photon correlation spectroscopy. The triglyceride (TG), phosphatidylcholine (PC), and penclomedine (in drug loaded emulsions) contents in each fraction were determined by HPLC. The aqueous-entrapped volume within Intralipid was determined to be approximately 10% by size-exclusion chromatography using [3H]mannitol. Thirteen sedFFF fractions collected from the drug free emulsions yielded particles ranging in size from 154 to 423 nm. Total channel recoveries were 89% and 95% for TG and PC, respectively. Apparent particle densities varied significantly with size, suggesting heterogeneity in composition as confirmed by PC/TG mass ratios which varied dramatically. Computer fits of the distribution profiles suggested populations of phospholipid vesicles and oil droplets containing excess phospholipid in addition to classical emulsion droplets. Drug loading induced a significant shift of the predominant triglyceride containing population to a larger particle size. The penclomedine distribution profile closely mimicked that of the TG rather than the PC fraction. These studies suggest the need to consider not only size distribution but also compositional distribution in characterizing parenteral emulsions.

Study Objective: The tuberculin skin test is the best diagnostic method to detect tuberculous infection. How accurate is interpretation of the test?, Design: Observational study., Setting: Both general hospital and university hospital., Participants: One hundred seven health-care professionals, including 52 practicing pediatricians, 33 pediatric house officers, 10 pediatric academicians, 11 registered nurses, and 1 pediatric nurse practitioner., Study: A tuberculin skin test (Mantoux) was applied to the arm of a known tuberculin converter. As participants entered/left the room, they were guided to the tuberculin converter. At no time did a participant observe readings other than his/her own., Results: Mantoux tuberculin reaction measuring 15 mm induration was read individually by a group of 52 practicing pediatricians, 33 pediatric house officers, 10 pediatric academicians, 11 registered nurses, and one pediatric nurse practitioner. The median induration recorded by this group of 107 health-care professionals was 10 mm, and 17 (33%) practicing pediatricians read the reaction as <10 mm induration. Using the > or = 15-mm induration indicator to identify a positive reaction, 93% of those in the study (99/107 participants) would have identified our known converter as tuberculin negative., Conclusion: This study confirms a general inaccuracy in interpretation of the tuberculin skin test reaction. It raises two questions. (1) Is there a general tendency toward underreading? (2) Does this general tendency to underread tuberculin skin test reactions raise some question as to the American Academy of Pediatrics, American Thoracic Society, and Centers for Disease Control and Prevention move in raising the amount of induration considered tuberculin positive to 15 mm in low-risk individuals?

We measured 13 metals in the urine of 496 United States residents to establish reference range concentrations using inductively coupled argon plasma mass spectrometry and Zeeman graphite furnace atomic absorption spectrometry. We frequently found 8 of these analytes at detectable concentrations in urine specimens: molybdenum (in 99.8%); lead (98.8%); tin (89%); thallium (77%); antimony (73.5%); manganese (73%); cesium (71%); tungsten (70%); and platinum (69.7%). The 95th percentile concentration for molybdenum was 168 micrograms/L; concentrations ranged up to 688 micrograms/L. Lead concentrations ranged up to 67 micrograms/L, and the 95th upper percentile was 6.4 micrograms/L. Tin had 95th upper percentile of 20.1 micrograms/L. Other analytes measured at detectable concentrations included barium (in 67% of the specimens); beryllium (67%); chromium (54%); thorium (44%); and cobalt (43%). In almost every case, the 95th upper percentiles of these analytes were less than 15 micrograms/L.

An extensive multi-laboratory study was conducted to compare three different field-flow fractionation (FFF) systems for use in the analysis of human erythrocytes. The object of this study was to determine the relationship between the FFF elution properties for each system and the traditional hematological blood cell parameters. One centrifugal system (Utah) and two gravitational systems (Paris and Abbott) were compared. In order to analyze erythrocyte populations with a broad range of hematological indices, blood samples were collected from individuals heterozygous for sickle cell anemia (A/S) and also from normal controls (A/A), and these were analyzed at each site. Identical samples were analyzed by the Abbott and Utah sites. With all three systems, blood samples from each category produced narrow, overlapping distributions of FFF retention ratios, with the Abbott and Utah systems showing slight elevations in the mean retention ratios for the sickle cell samples. Blood cell elution peak characteristics were compared with standard hematological parameters for each of the FFF systems, and negative correlations were consistently found between mean corpuscular volume (MCV) and retention ratios. Positive correlations were found between red cell distribution width (RDW) and retention ratios. Elevated FFF retention ratios were frequently found with blood samples having abnormal hematological profiles. These results demonstrate that the three differently configured systems all produce similar analysis profiles for erythrocytes from the classes studied here. The relationships between FFF parameters and hematological indices were consistent for all systems.

The binding of 5'-end biotinylated DNA, ranging in size from 100 to 5000 base pairs, was studied using streptavidin-coated polystyrene latex particles with diameters between 0.944 and 0.090 micron. The experimental binding constants and forward rate constants of this solid-phase reaction were determined to be several orders of magnitude lower than values for the biotin-streptavidin interaction in solution as expected and were shown to depend on the size of both ligand and substrate. An observed inflection in the binding constant of the biotinylated DNA appeared around 1000 base pairs, possibly indicating different surface orientations of the macroligand above and below this critical size. This effect was more pronounced for the smaller latex particles used in this study and highlighted possible differences in the surface arrangement of streptavidin on the differently sized particles. Diffusion limitation to the binding reaction was found to be significant in all cases. In this present work, an exponential relationship was established between the experimental binding constant and the number of base pairs in the biotinylated DNA. This relationship possibly provides a means to predict capacity and binding speed in cases where adsorption, purification, and release of larger DNA chains are required.

Human red blood cells were treated in different ways to alter their membrane deformability, and the hydrodynamic behavior of these altered cells was studied using the steric field-flow fractionation (FFF) technique. The relationships between cell retention in the FFF channel, flow-rate of the carrier fluid and the applied field strength were studied for normal and glutaraldehyde-fixed human red cells, and separation conditions were optimized. The effect of flow-induced hydrodynamic lift forces on red cell retention in the steric FFF channel was studied, and the results suggest that the membrane deformability of the red cell is an important factor contributing to the lift force, besides other previously described effects due to density and flow velocity. Using steric FFF, a mixture of normal and glutaraldehyde-fixed human red cells was completely separated with a resolution twice that found in published data from gel permeation, another hydrodynamic separation technique. Partial loss of membrane deformability, induced by different degrees of glutaraldehyde-fixation, by diamide, or by a thermal treatment, has also been studied. Steric FFF is thus shown to have potential for rapid separation and differentiation of red cells with different density and membrane deformability, conditions known to be associated with, e.g., cell senescence and certain hematological diseases.

Little is known about the control of events ending the lengthy prophase of meiosis I and leading to the G2/M-phase transition in mammalian spermatocytes, primarily because the relevant late pachytene, diplotene, and MI cells are present in low numbers in the testis and it is not possible to isolate them in significant numbers. We have utilized short-term cultures of pachytene spermatocytes from the mouse to study events of the G2/M cell-cycle transition induced by the protein phosphatase inhibitor okadaic acid (OA). Treatment of cultured pachytene spermatocytes with OA induced a rapid and premature onset of events leading to the M phase, visualized cytologically by nuclear envelope breakdown and chromosome condensation. After OA treatment, condensed chromosomes were seen as bivalents, not as univalents. Treatment with OA induced disassembly of synaptonemal complexes and resolution of crossovers as cytologically visible chiasmata. Chiasmata counts were similar in treated cells and control cells. Thus, surprisingly, even though the treated cells were in the pachytene substage of meiotic prophase, events of recombination were apparently completed to the point of chiasma formation in the majority of these cells. The sex chromosomes, forming the sex body of the pachytene spermatocyte, lagged behind the autosomal chromosomes in their condensation and progression toward the M phase. Treatment with OA induced an increase in histone H1 kinase activity, generally used as an indicator of metaphase-promoting factor (MPF) activity; furthermore, the OA-induced cell-cycle transition does not require new protein synthesis. These results suggest that OA treatment overrides a cell-cycle checkpoint control that normally keeps pachytene spermatocytes in a lengthy prophase and that this control may be exerted by regulation of protein phosphorylation status.

The binding of 5'-end biotinylated DNA fragments was compared between streptavidin (SA)-coated commercial M-280 magnetic latex particles with a diameter of 2.8 microns and adsorption-coated polystyrene (PS) latex standard particles whose diameter is 0.272 microns. Amino acid analysis showed the protein content of the commercial particles to correspond to 4x monolayer coverage, while the adsorption-coated PS particles displayed monolayer coverage, or 8 pmol/cm2. A fluorescence-based method was developed to quantify the adsorption of FITC-labeled SA, biotin, and biotinylated DNA. The validity of the method was substantiated for the labeled protein by both amino acid analysis and a colorimetric protein assay. While the specific binding of biotin was 0.38 mol per mole of SA on the adsorption-coated 0.272-microns particles and slightly higher (0.6 mol per mole SA) on the 2.8-microns particles, the specific binding of the bulky biotinylated 300-bp DNA was more favorable on the smaller particle (0.12 mol per mole SA for 0.272 microns versus 0.04 mol per mole SA for 2.8 microns).

In this paper, we demonstrate that by employing a combination of sedimentation field-flow fractionation (sedFFF) and other characterization techniques, such as photon correlation spectroscopy (PCS) and freeze-fracture electron microscopy (EM), it is possible to show that commercial fat emulsions of similar overall chemical compositions not only may exhibit different size distributions but may have different densities as well. A closer look at the density difference between droplet and suspension medium, on the one hand, and the droplet size, on the other, demonstrates that fat emulsions may have structures other than the traditional oil droplet surrounded by a monolayer of surfactant. From our determined and simulated density differences, we propose that these emulsion droplets may have a multilayered surfactant arrangement as well as an inclusion of water vesicles in the oil phase of the emulsion. Freeze-fracture EM observations provide evidence to confirm the existence of such complex structures. These findings are supported by recent EM work from other laboratories, as well as through chemical verification of elevated water contents in the oil droplets of these emulsions.

Sedimentation field-flow fractionation (SdFFF) has been used to characterize the adsorption of the proteins beta-casein (BCN) or beta-lactoglobulin (BLG) on colloidal polystyrene latices; this system was used to model hydrophobic interactions between the proteins and the surfaces of fat droplets in protein-stabilized emulsions. It was found that the SdFFF technique could determine directly the surface concentrations of BCN and BLG irreversibly adsorbed to the latex surface, provided care was taken to maintain the ionic strength of the carrier at a level which suppressed particle-wall repulsion in the separation channel. The measured surface concentrations were similar for the two proteins (about 1 mg/m2), and this was verified by quantitative amino acid analysis. These concentrations were smaller than those found in depletion studies (3 and 4 mg/m2 respectively for BCN and BLG), in which loosely associated protein may have been included in the determinations. The thickness of the adsorbed layers was determined in situ by dynamic light scattering and was found to differ significantly for the two proteins (up to 15 nm for BCN vs. 2-3 nm for BLG). The implication of these findings in terms of different surface arrangements of the two proteins is discussed.

The regulation of adenylyl cyclase activity by varying concentrations of Ca2+ was examined in plasma membrane preparations derived from a number of neural and non-neural cells. Enzyme activity in neural tissue (i.e. cerebellum) neural-derived pheochromocytoma PC12 cells and certain endocrine cells (i.e. pancreatic RINm5f and parathyroid cells) was stimulated by physiologic concentrations of Ca2+ by a calmodulin (CaM)-dependent mechanism. In contrast, adenylyl cyclase activity in non-neural cells (e.g. platelets and GH3 cells) was not stimulated by Ca2+. In these latter sources, enzyme activity was inhibited by increasing concentrations of Ca2+, independent of CaM. In liver membranes, Ca2+ and/or CaM did not alter adenylyl cyclase activity. These results demonstrate that the effects exerted by physiologic concentrations of Ca2+ on adenylyl cyclase activity range from CaM-dependent stimulation of activity to no effect, to CaM-independent inhibition of activity. The actions of Ca2+ on adenylyl cyclase may be major contributors to the various synergistic or antagonistic interactions that are seen between cAMP-generating and Ca(2+)-mobilizing systems.

Conventional operation of field-flow fractionation (FFF) systems involves carrying out the analysis at a constant flow of carrier; the flow is temporarily interrupted after injection of a sample in order to permit its equilibration under the applied field. Retention is calculated as the ratio of elution times for a non-retained species and the sample of interest, respectively. Such time-based retentions are only valid if the flow-rate is precisely known at all times during the run. The peristaltic pumps often used with FFF equipment are shown to have an output which varies unpredictably in time. Furthermore, initiation of flow after relaxation is shown to result in significant periods of transient behaviour while the system adjusts to the operating pressure. These and other variations in flow-rate can be eliminated as sources of error by basing the retention measurement on effluent weight, rather than on time. For this purpose, an electronic balance is interfaced with the system's computer, so that detector response/effluent weight data pairs are continuously monitored during the course of the FFF analysis.

A ten-component mixture of polystyrene latex particles in the 67-1220 nm size range was subjected to analysis by analytical ultracentrifugation (AUC) and sedimentation field-flow fractionation (SdFFF) using programmed and constant fields. The AUC analysis of the mixture yielded diameter values in good agreement with data determined on the separate components; the relative amounts of each component in the mixture were likewise closely reproducing the sample's known composition. Diameters determined by SdFFF, either in a constant- or programmed-field mode, were in good agreement with the AUC for particles smaller than about 500 nm. For the sample's larger components, however, particularly the programmed mode showed diameter values smaller than expected. In addition, field programming resulted in incomplete recoveries of the larger particles, leading to more or less distorted mass distributions for the complex sample. The observed discrepancies, which are thought to result from events at the analytical wall in the FFF channel, suggested a protocol for accurate sizing, as opposed to fingerprinting, of samples with broad size distribution. By tracking sizes and amounts of the different components at different but constant field strengths, and retaining as analytically valid only those data recorded in a retention range from five to about thirty column volumes, it was possible to determine sizes and amounts in good agreement with known parameters for the sample. Unlike the AUC procedure, SdFFF produces fractions of a high degree of uniformity, which lend themselves to a secondary analysis, e.g. by electron microscopy, as shown in the study.

The GTP-dependence for stimulatory and inhibitory regulation of plasma membrane adenylate cyclase activity was measured in plasma membrane fractions isolated from a variety of cell types (platelets, lymphocytes, PC12 cells, GH3 cells, NBP2 cells, and hepatocytes). This report shows that the isolation of plasma membranes for the study of GTP-dependent adenylate cyclase activity was, for some cells, enhanced by the exposure of the cells to glycerol prior to cell lysis. The isolation of plasma membranes from other cells, which did not appear to be sensitive to glycerol pretreatment, was enhanced by the removal of heavy particulate matter prior to fractionation of the cell lysate. The regulation of enzyme activity by various agents was found to be dependent upon the presence of (exogenous) GTP to varying degrees, indicating variable contamination of membrane preparations with GTP. It is concluded that (i) exposure of platelets and lymphocytes to glycerol prior to cell lysis decreases subsequent contamination of the plasma membrane preparation with GTP, and (ii) although glycerol pretreatment of other cells does not ensure the subsequent isolation of plasma membrane adenylate cyclase activity displaying high requirements for (exogenous) GTP, it is a reasonable first approach to be used during the development of procedures for the isolation of plasma membranes.

It is shown that the method known as sedimentation field-flow fractionation, which has been applied to the separation and characterization of many industrial and biological particles and recently to emulsions, can be used to obtain high-resolution droplet diameter profiles for perfluorocarbon blood substitutes. Following a description of the methodology, experiments are described for two commercial perfluorocarbon emulsions, Fluosol-DA 20% and Fluosol-43. The droplet diameter profiles for both of these blood substitutes are shown to shift to noticeably higher diameter values in less than 2 months. The diameter at the profile peak for Fluosol-DA 20%, for example, shifts from 0.19 to 0.27 micron in 56 days.

Sedimentation field-flow fractionation (FFF) is a new technique that separates and characterizes submicron particles. In the present work, two independent sedimentation FFF methods are presented to characterize bovine serum albumin microspheres in terms of particle size, polydispersity, and diffusion coefficient. Particle diameters and polydispersities determined by the two sedimentation FFF methods were in excellent agreement with each other and in good agreement with values calculated from transmission electron microscopy (TEM) measurements. The diameters calculated from the two FFF methods and TEM were 0.349, 0.346, and 0.354 microgram, respectively.

Lead interferes with heme synthesis in erythrocytes and has a deleterious effect on red cell membranes. We measured myeloperoxidase (MPO) enzyme activity in the granulocytes of dogs fed increasing quantities of lead. Concurrently, iodination capability and in vitro bactericidal activity were measured. Blood lead levels were monitored. Three of 4 dogs poisoned with lead developed significant decreases in MPO enzyme activity in their granulocytes. The decline in MPO activity correlated with cumulative lead toxicity as judged by blood lead levels and clinical signs of lead poisoning. Iodination ability in all 4 dogs decreased with cumulative lead toxicity. After discontinuation of lead administration, recovery of granulocyte MPO activity preceded recovery of iodination ability. This observation suggests the possibility of separate effects of lead on iodination ability and MPO activity. Moderate impairment of bactericidal capacity developed in 3 of 4 dogs with severe lead poisoning. Clinical infections were not observed during the course of the study.

The enzymatic hydrolysis of whole gliadin has been studied in vitro using sequential treatments by pepsin, trypsin, and pancreatin. Amino-terminal pyroglutamic acid-peptides were formed at each stage of the digestion process and the concentration of these peptides increased as the hydrolysis proceeded. Digests were further fractionated on columns of AG 50W-X8 or SE-Sephadex. Enzymic digests and selected column fractions were analyzed with pyrrolidonecarboxylate peptidase. Each digest or fraction was degraded further by this peptidase. Enzyme activity was greatest towards peptic-tryptic-pancreatic digests, peptic-tryptic digests, peptic digests, and undigested gliadin, in that order. The stability of lysosomal membranes to synthetic L-pyroglutamic acid, L-pyroglutamyl-L-alanine, L-pyroglutamyl-L-proline, and to selected fractions of the enzymic digests was tested. Each treatment ruptured lysosomal membranes. Findings are discussed in relation to the normal catabolism of gliadin and the alterations that may occur in certain pathological states.

Sedimentation field flow fractionation (sed-FFF) is shown to be a valuable procedure for analysis of a wide variety of subcellular particle preparations. The principles underlying this relatively new separation procedure are described. Separation is based on differences between particles in mass and/or density. As in chromatography, the procedure involves relating on-line or off-line measurements made on the effluent from the separation chamber to the elution (retention) time. In this work effluents were monitored for absorbance at 254, 280, and/or 320 nm; collected fractions were assayed for protein content, total ATPase activity, and/or marker enzyme activities and, when appropriate, were examined by electron microscopy. The ratio of the absorbances at 254 and 320 nm was found to provide a sensitive measure of partial resolution of subcellular particles. Preparations containing all of the subcellular particles of corn roots (exclusive of nuclei, cell walls, and ribosomes), and fractions thereof enriched in mitochondria, microsomes, Golgi membranes, or plasma membranes, were examined by sed-FFF. The subcellular particles appear to remain largely intact. All of the particles observed had a mass less than 2 X 10(11) g/mol. All of the preparations were grossly heterogeneous with respect to effective mass distribution. This is due in part to heterogeneity with respect to the organelle of origin. In microsome preparations, components of low, medium, and high density were present in the unretained peak; the retained region had comparatively more high density particles. Plasma membrane preparations had a very wide effective particle mass distribution. The observations suggest that, in addition to its utility for analytic purposes, sed-FFF is likely to prove useful for micro-preparative fractionation of some subcellular particle preparations. Sed-FFF and density gradient centrifugation can be utilized as complementary methods.