Antigen-specific electrophoretic cell separation for immunological investigations

Preincubation of human blood lymphocytes with cell surface antigen specific antibodies under non-capping conditions reduces the electrophoretic mobility of the corresponding lymphocyte subpopulation. Antigen-positive and antigen-negative cells can be separated by free flow electrophoresis with high yield, purity and viability. The use of fluorescence-labelled second antibodies augments the induced decrease in net surface charge density, and allows rapid detection of antigen-positive cells in the fractions of electrophoresis. Carrier-free cell electrophoresis of human peripheral blood lymphocytes after reaction with anti-IgM-antibody or the monoclonal antibodies OKT4 or OKT8, and sandwich staining with tetrarhodamine isothiocyanate-labelled anti-IgG resulted in the large-scale separation of high pure human B and T lymphocyte subpopulations. Their functional integrity was shown in assays of lymphocyte transformation and of antigen-specific induction and regulation of antibody synthesis in vitro. These separate lymphocyte subpopulations are useful tools for immunological investigations. While, for instance, the effects of drugs on human lymphocytes are obscured by coincident changes in cell composition of the peripheral blood tested that do not by themselves reflect whole body immunocompetence, the cell separation and in vitro assays at a defined cell number and cell composition allow the recording of quantitative changes in the function of different cell subpopulations. We studied the influence of the anesthetic thiopental on separated human lymphocyte subsets. In both polyclonal lectin stimulation and in vitro antibody production, thiopental exhibited a noncytotoxic suppression of lymphocyte functions. B-Cells, T-helper and T-suppressor cells were equally affected and showed the same dose response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational changes in poly(vinyl alcohol)-graft-polyacrylamide in aqueous solutions vs graft content

Conformational changes have been studied in intramolecular polymer-polymer complexes (intraPC) of graft copolymers of poly(acrylamide) and poly(vinyl alcohol) (PVA-g-PAA) with various numbers of grafts (4-42) per molecule as a function of temperature and copolymer concentration. It is shown that the magnitude of conformational change depends on the grafts content while the temperature range over which the conformation changes occur is essentially determined by copolymer concentration. The conformational changes are reversible on heating and cooling.     

pH-induced reversible conformational and morphological regulation of polyleucine grafted polyallylamine assembly in solution

One of the essential parts in the molecular mechanism of biological properties is the structural changes of proteins induced by stimuli. An amphiphilic copolymer, poly(L-leucine) grafted polyallylamine as a simple model of proteins, has been prepared by NCA polymerization with free amino groups of polyallylamine as an initiator. Here, we report the pH-induced reversible conformational and morphological regulation of the amphiphilic copolymer, whose hydrophobic peptide graft chains have no pH-sensitive groups, in an aqueous solution containing 50 vol % trifluoroethanol. The conformation of the poly(L-leucine) graft chain was found to be strongly pH dependent. Under acidic conditions, where electrostatic repulsion existed between the neighboring protonated amine moieties of the polyallylamine main chain, the rapid aggregation of the poly(l-leucine) graft chains was disturbed, and the peptide graft chains formed a beta-sheet structure owing to the intramolecular hydrogen bonding among the graft chains. Under this condition, the amphiphilic polymer formed amyloid-like fibrils, and then the fibrils grew into a planer plate composed of staked beta-sheets. On the other hand, under basic conditions, the poly(L-leucine) graft chains showed conformational transitions from a beta-sheet structure to an alpha-helical conformation owing to a distortion of the regular arrangement of the peptide graft chains by the conformational change of the polyallylamine main chain, whose amino groups were deprotonated. The conformational transition resulted in a disturbance of the regular sheet assembly of the amphiphilic copolymer and induced morphological changes to the amorphous globular aggregates. The pH-induced conformational and morphological changes of the poly(L-leucine) graft polyallylamine were reversible and synchronized with the protonation of the polyallylamine main chain.     

Temperature‐induced conformational transition of bovine serum albumin in neutral aqueous solution by reversed‐phase liquid chromatography

The temperature‐induced conformational transition of bovine serum albumin (BSA) in neutral aqueous solution was studied using intrinsic fluorescence emission spectrum, reversed‐phase liquid chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the conformation transition thermodynamic parameters were determined in the temperature range 12–50 °C. The results showed that, in the temperature range 12–20 °C, BSA only existed in a single conformation state A, while in the temperature range 22–50 °C, it existed in two different conformation states: A and B. The percentage of conformation state A decreased while that of conformation state B increased with the increase in temperatures, and when temperature approached 50 °C conformation state B accounted for approximately 25% of all conformation states of BSA. In the conformational transition of BSA from conformation state A to conformation state B, the positive enthalpy change, entropy change and free energy changes demonstrated that the conformational transition was endothermic, nonspontaneous and mainly entropy‐driven. © 2013 The Authors. Biomedical Chromatography published by John Wiley & Sons, Ltd.     

Kinetics of Complexation in the Monolayers of Amphiphilic Dicetylcyclene on the Surface of Aqueous Copper(II) Chloride Solutions

The kinetics of complexation in monolayers of dicetycyclene at the surface of aqueous copper(II) chloride solutions was studied. It was shown that the changes in the phase state of monolayer related to the conformational transitions of macrocycle are responsible for the differences in the rate and binding mechanism of copper ions. It was concluded that the sterically more advantageous (for the coordination with the metal ion) conformation of macrocyclic polyamine is ensured in a monolayer. In addition, it was established that the rate and mechanism of complexation in such monolayers greatly depend on the degree of protonation of ligands, the latter being dependent on the subphase pH. It was also demonstrated that an increase in subphase pH to 7 and higher results in an almost total suppression of metal ion binding due to strong conformational distortion of dicetylcyclene macrocycles in a monolayer and the hydrogen bonding between macrocycles.     

Effect of flow and surface conditions on human lymphocyte isolation using microfluidic chambers

Phenotypically pure subpopulations of lymphocytes can provide valuable insights into the immune response to injury and disease. The isolation of these subpopulations presents unique challenges, particularly when preprocessing incubation to attach fluorescent or antibody tags is to be minimized. This paper examines the separation of T and B lymphocytes from mixtures using microfluidic chambers coated with antibodies, focusing on flow conditions and surface chemistry. The adhesion of both cell types decreases as shear stress increases irrespective of the surface chemistry. The incorporation of poly(ethylene glycol) chains along with the antibodies on the chamber surface is shown to significantly improve the reproducibility of cell adhesion and is thus an important part of the overall system design. Furthermore, this technique is shown to be an effective way of isolating highly pure subpopulations of lymphocytes from model mixtures, even when the target cell concentration is low.     

Conformational Switching of Resorcin[4]arene Cavitands by Protonation,Preliminary Communication

The synthesis of the quinoxaline‐bridged resorcin[4]arene cavitand 1 was accomplished from 2‐[3,5‐di(tert‐butyl)phenyl]acetaldehyde via formation of the intermediate octol 2 . Such cavitands are known to occur in an open `kite' conformation at low temperature (<213 K) but to adopt a `vase' conformation at elevated temperatures (>318 K). We discovered that protonation of cavitand 1 at room temperature by common acids, such as CF₃COOH, also causes reversible switching from `vase' to `kite', and that this conformational change can be conveniently monitored by both ¹H‐NMR and UV/VIS spectroscopy.     

The effect of suntan parlor exposure on delayed and contact hypersensitivity

Cutaneous and systemic immune function are believed to play an important role in cutaneous carcinogenesis. We therefore sought to determine whether the suntan parlor radiation sources commonly used in the United States cause measurable qualitative suppression of immune function and quantitative alterations in circulating T cell subpopulations. Subjects (n = 22) were recruited and randomly assigned to receive suntan parlor exposures (10 full-body UV exposures over a 2 week period, shielding only the right flexural arm) or no exposure. Baseline circulating T lymphocyte subpopulations (T helper lymphocyte, CD4; T suppressor/cytotoxic lymphocyte, CD8) were measured. Two weeks later (upon completion of UV exposures for those in this group), circulating T cell subpopulations were measured and dinitrochlorobenzene (DNCB) sensitization (in the UV group, on the UV-exposed buttock) was performed. Subsequent DNCB elicitation was performed in a bilateral fashion (in the UV group, on the right UV-shielded and the left UV-exposed upper arm). We found that subjects in the UV group demonstrated localized suppression of contact hypersensitivity sensitization and elicitation and also an increase in circulating CD8 cells when compared to the control group (P < or = 0.05). We conclude that suntan parlor exposures, as typically received in this country, suppress contact hypersensitivity and increase the circulating T suppressor/cytotoxic cell number quantity.     

Molecular mechanics study on conformation of perylene-quinonoid photosensitizers

Using molecular mechanics method,values of the heat of formation (HF) of different conformations,of perylenequinonoid photosensitizes hypocrellin A (HA) and hypocrellin B (HB) were calculated and the variance of HF after phenolic protons' dissociation were calculated as well The following was found:(i) The HF values of lour conformational isomers of HA and HB are similar to each other,so the four isomcrs can transform to each other room temperature,(ii) There exists the difference between the ability of dissociation of phenolic protons of HA and that of HB,the former is higher than the latter (iii) There exist two intramolecular hydrogen bonds in HA and HB The bond energy is approximately 8 kJ/mol and the energy of conformation Ⅰ is lower than that of conformationⅡ The bond energy of HA is lower than that of HB.(iv) There exists a low energy snot when phenolic hydroxyl bond twists 180° from the position where hydrogen bond is formed,which suggests that this kind of conformation probably exists,(v) Th     

电子辐照及再结晶P(VDF-TrFE)共聚薄膜红外光谱研究

利用傅立叶变换红外光谱(FT-IR)研究聚偏氟乙烯与聚三氟乙烯共聚薄膜[P(VDF0.80-TrFE0.20)]的电子辐照和再结晶处理过程中分子链构型变化和化学变化, 为深入探讨辐照的改性机理提供依据. 研究发现, 辐照后薄膜分子链全反构型百分含量随吸收剂量增大而迅速减少, TG构型和T3G构型百分含量显著增多. 而当吸收达一定剂量时, 三种构型相对含量基本不再变化, 表明高剂量辐照时样品极性回升不依赖分子链构型中全反型的含量的增多, 而是和高交联度的边界效应有关. 再结晶过程中分子链构型变化恰好和辐照效应产生的变化相反, 并且形成了更加稳定的C=C共轭结构.     

Three models for chain conformation of block copolymers in solution and in solid state

Solution properties of polystyrene-poly(methyl methacrylate) (PS-PMMA) diblock copolymer, polystyrene-poly(tertiobutyl methacrylate) (PS-PtBuMA) diblock copolymer, and poly(ethylene oxide)-polystyrene-poly(ethylene oxide) (PEO-PS-PEO) triblock copolymer have been measured by viscometry. The PEO-PS-PEO copolymer has been studied also in solid state by differential scanning calorimetry and by optical microscopy. All the block copolymers present a conformational transition in solution at a given temperature region which is relatively narrow. If below this transition temperature a copolymer adopts a segregated conformation (dumb-bell model), above this transition adopts a nonsegregated or pseudo-gaussian conformation, and vice versa. In the transition temperature region the copolymer adopts a compressed segregated conformation (core and shell model). If the passage from the solution to the solid state is performed in a given constant temperature in which the copolymer presents a segregated or nonsegregated conformation the same conformation is observed in the solid state (memory effect). © 1996 John Wiley & Sons, Inc.     

Mitogenic activity of Tulipa gesneriana lectins on mouse and human lymphocytes.

Tulipa gesneriana lectin-erythrocyte (TGL-E) which agglutinates mouse erythrocytes showed a potent mitogenic activity on mouse spleen cells and human peripheral blood lymphocytes, however, TGL-E had only slight mitogenic activity on mouse thymus cells. Its subunit alpha with a molecular weight (MW) of about 26,000 showed a potent mitogenic activity as did that of native lectin, but subunit beta with a MW of about 14,000 showed no activity, indicating that the mitogenic activity of TGL-E originates from subunit alpha. TGL-E stimulated T cell enriched spleen cells which passed through a nylon column, but not spleen cells from a nude mouse or spleen cells treated with anti-Thy 1.2 antibody and complement. Thus, TGL-E stimulates only mouse T cells but not B cells. The other lectin in tulip bulbs, Tulipa gesneriana lectin-yeast showed no mitogenic activity on mouse spleen, thymus cells or human paripheral blood lymphocytes.     

Independence of photoproduct formation on DNA conformation.

Abstract— In an ethanolic solution native T7 DNA can undergo conformational transitions from the B conformation (0% ethanol) to the C-like (60% w/w ethanol) and the A (80% w/w ethanol) conformations. We have investigated the formation of three classes of thymine-derived photoproducts in T7 DNA irradiated (280 nm) in the B, C-like, and A conformations, which were monitored by circular dichroism measurements. We find that the predominant class of thymine-derived photoproducts in any conformational state is cyclobutyl dipyrimidines. While the ‘spore product,’ 5-thyminyl-5,6-dihydrothymine, which belongs to another class of photoproductsf does form in native DNA in the A conformation, its yield in denatured DNA at 80% ethanol is the same as that in native DNA. The yield of pyrimidine adduct, a third photoproduct class, is a maximum at 50–60% ethanol. This effect of ethanol is probably not due to the ethanol-induced C-like conformation, however, since pyrimidine adduct formation is not enhanced when T7 DNA is irradiated in the C conformation in 6 M CsCl or in intact phage. We conclude from these and other data in the literature that the degree of hydration rather than the conformational state is the critical factor in determining which of the photoproducts will form in native DNA.     

AM1 molecular orbital studies of the structures,conformations, protonation energies,and electronic properties of triazine dihydrofolate reductase inhibitors

The structural, conformational, and electronic properties of three triazine antifolates were determined by AM1 molecular orbital calculations, and the results were compared with other theoretical studies and with X-ray crystallographic studies of these and similar triazines both in the crystalline state and as complexes bound to dihydrofolate reductase. Calculated protonation energies confirm crystal structure data indicating N-protonation analogous to that reported for MTX in similar environments. Overall, the calculated structural and conformational properties are in good agreement with X-ray crystallographic results for these and similar triazines as found in the crystalline state and in enzymebound ternary complexes. However, for one triazine AM1 predicts a conformation with the bulky aromatic substituent twisted about 60° away from coplanarity with the triazine ring, in contrast to the nearly coplanar conformation found in the crystalline state. Intermolecular interactions favoring the coplanar conformation may thus be operative in the crystalline environment. The unique conformational preferences and greater conformation flexibility of triazines in general and of this triazine in particular may provide a key to understanding their biological activity.     

Modulation of Conformational Preferences of Heteroaromatic Ethers and Amides through Protonation and Ionization: Charge Effect

Multiple approaches reveal the strong effects of a positive charge introduced by protonation or ionization on the conformation of o-heteroaromatic ethers and amides. The ethers and amides containing an ortho-N heteroatom are syn-preferring while those containing an ortho-O or ortho-S heteroatom are mostly anti-preferring. However, for all the monocyclic o-heteroaromatic ethers and amides, the protonated ones are all anti-preferring while the ionized ones are all syn-preferring. Interestingly, although both the protonation and ionization introduce a positive charge, they have such different effects on molecular conformation, very informative for understanding the origin of conformational preferences. Detailed analysis shows that the population of the introduced positive charge dictates the conformational preferences via electrostatic and orbital interactions. Compared to ortho-heteroatoms, meta-heteroatoms have weaker effect on conformational preference. Achieved by complete inductive method, the regularity of conformational preferences and switching provides easy ways to modulate conformers (by pH or redox), and makes this kind of ether or amide bond a conformational hinge applicable to design of functional molecules (drugs and materials) and modulation of molecular biological processes.     

Human lymphocyte sorting by gravitational field-flow fractionation

Interest in biological studies on various cell types for many biomedical applications, from research to patient treatments, is constantly increasing. The ability to discriminate (sort) and/or quantify distinct subpopulations of cells has become increasingly important. For instance, not only detection but also the highest depletion of neoplastic cells from normal cells is an important requisite in the autologous transplantation of lymphocytes for blood cancer treatments. In this work, gravitational field-flow fractionation (GrFFF) is shown to be effective for sorting a heterogeneous mixture of human, living lymphocytes constituted of neoplastic B cells from a Burkitt lymphoma cell line and healthy T and B lymphocytes from blood samples. GrFFF does not require the use of fluorescent immunotags for sorting cells, and the sorted cells can be collected for their further characterization. Flow cytometry was used to assess the viability of the cells collected, and to evaluate the cell fractionation achieved. A low amount of neoplastic B lymphocytes (less than 2%) was found in a specific fraction obtained by GrFFF. The high depletion from neoplastic cells (more than 98%) was confirmed by a clonogenicity test.     

Conformational Switches: Controlling the Carbamate C-N Rotamer Equilibrium

The syn / anti rotamer ratio for tert -butyl N -(2-thiazyl)carbamate 3 was measured by solution-state NMR in the presence and absence of a donor-acceptor-donor hydrogen bonding template. The template is able to switch the conformation of carbamate 3 from >95% anti to >70% syn making it the most effective carbamate conformational switch yet reported. B3LYP calculations provide insight into the factors that produce this result.     

pH-dependent conformational switching in 2,6-benzamidodiphenylacetylenes

The conformational equilibrium of a pH-dependent switch based on an intramolecularly H-bonded diphenylacetylene can be predictably biased by using electron-donating or -withdrawing groups. Furthermore, protonation of the electron-donating dimethylamino group converts it into an electron-withdrawing dimethylammonium cation with a concomitant switch in conformation.     

Bis-4,4′-biphenyl Ring Embedded Octaphyrin with Three Distinct Conformational Structures

Three distinct conformational structures of carbaoctaphyrins were prepared by incorporating bis-4,4'-biphenyl units in the macrocyclic core. The free-base form adopts a figure-eight conformation, whereas the protonation triggers a conformational change with a pyrrole ring inversion and acquires an open-framework structure. The insertion of bis-Rh^I metal ion in the macrocyclic core affords a singly twisted conformational structure. Furthermore, the local aromaticity in the bis-4,4'-biphenyl ring dominates the overall macrocyclic aromaticity in all three forms, and thus adopts nonaromatic characteristics. These results are supported by spectral as well as theoretical studies, and they are unambiguously confirmed by X-ray crystal analyses.     

Estimation of cell surface associated protease activity and its application to lymphocytes.

A new method has been developed to estimate proteolytic activity available at the cell surface. Radioiodinated protein substrates are covalently linked to modified polystyrene-divinylbenzene beads with various diameters. These beads are presented to viable cells. Secreted enzyme activity is estimated when no contact occurs between beads and cells. Surface associated proteolytic activity is estimated by the increased rate of iodinated peptide release due to a contact between beads and cells. This method was applied to various lymphocyte preparations. In the absence of serum, mouse spleen lymphocytes produce three- to fourfold higher proteolytic activity than lymph node cells. This activity is completely inhibited by serum diluted 1:10. Since the proteolysis is so marked in the case of spleen cells, one must conclude that lymphocytes removed from the serum and treated in buffered mediums at 37 degrees C have enzymatically altered surface properties. Cell surface associated enzyme activity was measured using rat lymph node lymphocytes with less than 0.1% contamination by granulocytes. This predominantly thymus derived, T cell population had 30% increase in proteolysis due to contact between cells and solid-phase localized substrate of casein. The released enzymatic activity was inhibited by diisopropylfluorophosphate, but its effect on the surface associated enzyme activity remains questionable since it perturbs several membrane functions.