Magnetic properties and oxygen migration induced resistive switching effect in Y substituted multiferroic bismuth ferrite.

Multiferroic Y-doped BiFeO3 (BY10FO) thin films were deposited on FTO coated glass substrate using sol-gel spin coating technique. Y-doping causes structural distortion without changing the structure of the parent BiFeO3 (rhombohedral: R3c). The M-H hysteresis curve reveals that the BY10FO film exhibits saturation magnetization at a low-coercive field by suppressing the spiral spin modulated structure. The bipolar resistive switching behavior has been investigated on a Ag/BY10FO/FTO hetero-structure through conventional I-V curve measurements and the device can produce an ON/OFF ratio of around 12 over 30 complete testing cycles. The space charge limited current and Schottky barrier emission conduction mechanism play a crucial role in switching the states between HRS and LRS. The impedance spectroscopy analysis at HRS and LRS confirms the significant degradation of resistance from MΩ to kΩ. The switching mechanism in the hetero-structure is due to migration and recombination of oxygen vacancies present in the film. The non-degradation of the Ag/BY10FO/FTO device after several testing cycles confirms that the switching of resistance between ON and OFF states is reproducible, reversible and controllable to be used for possible future non-volatile resistive random access memory application.

Development of a western blot method for detection of fish ectoparasite Argulus siamensis antigens.

A study was undertaken to develop a western blot method for detection of immunogenic proteins of fish ectoparasite, Argulus siamensis for its further use as potential vaccine candidates. Argulus antigens were prepared by homogenization and injected to rohu (Labeo rohita) juveniles for development of immune serum. The serum was used to immunostain the antigens in western blot. The other reagents added in sequence were rabbit anti-rohu serum, goat anti-rabbit ALP conjugate and substrate (BCIP-NBT). However, similar banding patterns were observed with both control and immunized rohu serum. Hence, a possible cross-reaction was suspected and verified in number of western blot experiments. A typical cross-reaction observed was of rabbit serum reacting directly with Argulus antigens. Hence, the rabbit anti-rohu serum was replaced with guinea pig anti-rohu serum. Another cross-reaction of goat anti-guinea pig ALP conjugate with rohu serum was eliminated by using goat anti-rabbit ALP conjugate with guinea pig serum. Thus, the final western blot method consisting of Argulus antigens → rohu serum → guinea pig anti-rohu serum → goat anti-rabbit ALP conjugate → substrate, yielded distinguishing results between control and Argulus-immunized rohu serum samples. The developed test has tremendous downstream applications, particularly in immunoproteomic studies of Argulus antigens.

Modulation in the acidity constant of acridine dye with cucurbiturils: stimuli-responsive pKa tuning and dye relocation into live cells.

The noncovalent host-guest interactions of the cationic (AcH+) and neutral (Ac) forms of an acridine dye with macrocyclic hosts such as cucurbit[7]uril (CB7) and cucurbit[8]uril (CB8) have been investigated to evaluate the effect of cavity size on the photophysical properties and the protolytic equilibrium of the acridine dye. The cationic form undergoes significant complexation with CB7 (Keq = 106 M-1), causing a sharp decrease in the fluorescence intensity, whereas the neutral Ac form of the dye undergoes weak complexation with CB7 (Keq = 103 M-1) and the binding constant is lowered by three orders of magnitude compared to that of the CB7-AcH+ system. The Job plot revealed that both forms form a 1 : 1 complex with CB7. On the other hand, the AcH+ form shows strong emission quenching on interaction with CB8 and the formation of the 1 : 2 CB8 : AcH+ complex has been confirmed from the Job plot. The strong affinity of CB7 and CB8 to the protonated form resulted in a large upward pKa shift (ΔpKa ∼ 3.4 units for CB7 and ∼1.3 units for CB8) in the dye. Taking advantage of the above modulations in the fluorescence and pKa values, adamantylamine-induced fluorescence regeneration, controlled pKa tuning and dye relocation from the CB7 cavity to cell lines have been established for the first time, which find potential applications in fluorescence off-on sensing and drug delivery.

Metal ion-induced supramolecular pKa tuning and fluorescence regeneration of a p-sulfonatocalixarene encapsulated neutral red dye.

The host-guest interactions and the consequent modulation in the prototropic equilibrium of a phenazine dye, neutral red, with p-sulfonatocalix[4]arene (SCX4) and p-sulfonatocalix[6]arene (SCX6) macrocyclic hosts have been investigated. Both the neutral (NR) and cationic (NRH+) forms of dyes formed inclusion complexes with SCX6, with a larger binding constant for the latter (K = 8.6 × 105 M-1versus 4.8 × 103 M-1) due to the cation receptor behavior of the calixarenes. The distinct differences in the binding constant of NR and NRH+ provided a finite tuning of pKa between 6.5 and 8.8, through a competitive binding with metal ions. Importantly, the fluorescence quenching observed in the SCX-neutral red interactions stands in contrast to the fluorescence enhancement observed with other macrocyclic hosts, such as ß-cyclodextrin and cucurbit[7]uril. This is due to the unique portal stacking interaction of NRH+ with the SCXs, compared to the axial inclusion geometry documented for the other macrocycles. The electron transfer from the SCX to the neutral red dye is adjudged to be the effective excited-state relaxation pathway leading to fluorescence quenching. In combination with the metal-ion induced fluorescence regeneration and tuning the pKa value, the SCX-neutral red system finds potential applications in drug delivery, photodynamic therapy, catalysis, and sensor applications.

Cyclodextrin-assisted modulation of the photophysical properties and acidity constant of pyrene-armed calix[4]arene.

The supramolecular interaction of cyclodextrin homologues with a pyrene-armed calixarene has been investigated. p-tert-Butylcalix[4]arene-di-4-(pyren-1-yl)butanoic acid (PCX4) has been synthesized and characterized by 1H NMR, FT-IR and mass spectrometry. The modulation in the photophysical properties, especially the monomer and excimer emission behaviour of the pyrene group in PCX4 with ß-cyclodextrin (ß-CD) and γ-cyclodextrin (γ-CD), has been explored in aqueous solutions by using optical absorption, steady-state and time-resolved fluorescence measurements. Steady-state fluorescence studies illustrate that upon gradual addition of γ-CD to the PCX4 solution, initially there is a decrease in the monomer emission (at 378, 398 and 420 nm) along with the appearance of weak excimer emission at 482 nm. Beyond a particular concentration of γ-CD (>1.2 mM), PCX4 shows strong excimer emission along with an increase in the monomer emission, indicating two types of interaction modes between PCX4 and γ-CD. Fluorescence anisotropy measurements confirm intramolecular excimer formation with a lower concentration of γ-CD, whereas it predominantly becomes an intermolecular excimer beyond 1.2 mM of γ-CD. On the other hand, the monomer emission of the pyrene group of PCX4 increases in the presence of ß-cyclodextrin having lower cavity sizes due to the formation of a 1 : 1 complex. For the first time, the intrinsic fluorescence from the pyrene moiety of PCX4 is employed to determine the pKa value of PCX4 and it is ∼6.1. The cyclodextrin-encapsulated PCX4 brings a 1.2 unit downward pKa shift. Along with the pH-responsive interconversion of monomer to excimer emission of γ-CD complexed PCX4, this system can find applications in ratiometric sensing and optical supramolecular pH-sensors.

Breakdown of the x-ray resonant magnetic scattering signal during intense pulses of extreme ultraviolet free-electron-laser radiation.

We present results of single-shot resonant magnetic scattering experiments of Co/Pt multilayer systems using 100 fs long ultraintense pulses from an extreme ultraviolet (XUV) free-electron laser. An x-ray-induced breakdown of the resonant magnetic scattering channel during the pulse duration is observed at fluences of 5 J/cm(2). Simultaneously, the speckle contrast of the high-fluence scattering pattern is significantly reduced. We performed simulations of the nonequilibrium evolution of the Co/Pt multilayer system during the XUV pulse duration. We find that the electronic state of the sample is strongly perturbed during the first few femtoseconds of exposure leading to an ultrafast quenching of the resonant magnetic scattering mechanism.

Characterization of serum immunoglobulin in channa striata (BLOCH) and kinetics of its response to Aeromonas hydrophila antigen.

The immunoglobulin (Ig) from the serum of Channa striata was isolated by gel electroelution and characterized further to understand its nature and subsequent applications in studying the immune response. The purity of the sample was confirmed with the presence of a single band on native gradient PAGE and the molecular weight of âˆ¼897 kDa was determined from the gel. In SDS-PAGE, C. striata Ig was reduced to produce two bands corresponding to H (heavy) (∼72 kDa) and L (light) (∼27 kDa) chain subunits. Polyclonal antiserum against the purified Ig was raised in a rabbit and adsorbed with 10% liver tissue homogenate of C. striata to enhance its specificity. By an indirect ELISA standardized using the adsorbed rabbit antiserum, the normal serum Ig concentration in C. striata was estimated to be 3.48 mg/mL. Further, a kinetic study of specific immunoglobulin response to formalin-killed Aeromonas hydrophila antigen was undertaken using another indirect ELISA, which showed a significant increase in serum immunoglobulin titer from day 2 onwards and reached its peak at day 14. Subsequently, the Ig titer was dropped from day 21 onwards till the completion of the experiment at day 42, although it was at a significantly higher level than the control.

An assessment of heavy metal contamination in soils of fresh water aquifer system and evaluation of eco-toxicity by lithogenic implications.

The chemistry of heavy metals in sediments with respect to bio-availability and chemical reactivity is regulated by pH, texture, and organic matter contents of the sediments and specific binding form and coupled reactivity of the metals within. To focus on the metal distribution (Fe, Mn, Pb, Cd, Zn, Co, Cu, and Cr) and behavior in a fresh water aquifer system along with the ecological toxicity parameters, a four-step sequential extraction method was applied on 18 Eastern Ghats' type sediments from fluorosis-hit Nayagarh district, India. Geo-accumulation index of metals in the sediments indicates that they are practically uncontaminated and/or less contaminated with and Fe, Mn, and Cu; contaminated to moderately contaminated with Pb, Zn, and Cr; and strongly contaminated with Cd. Rather, more than 80 % recovered Cd metal concentration in sediments constitute the labile fractions. Temporal clustering of metal fractions indicates transition metal fraction distribution claiming the sediment pH regulation. Similarly, base metal distribution accounts for organic carbon and soil conductivity due to their greater availability in exchangeable and sulfide fractions. Correlation analysis and factor analysis scores demonstrate lack of inter-relationship between transition group and base metal fractions. High fluoride concentration in ground water is associated with high sodium-bicarbonate-iron affinity with elevated pH values (i.e., >7.0) and high positive factor score with the total iron concentration in ground water.

Holographically aided iterative phase retrieval.

Fourier transform holography (FTH) is a noise-resistant imaging technique which allows for nanometer spatial resolution x-ray imaging, where the inclusion of a small reference scattering object provides the otherwise missing phase information. With FTH, one normally requires a considerable distance between the sample and the reference to ensure spatial separation of the reconstruction and its autocorrelation. We demonstrate however that this requirement can be omitted at the small cost of iteratively separating the reconstruction and autocorrelation. In doing so, the photon efficiency of FTH can be increased due to a smaller illumination area, and we show how the presence of the reference prevents the non-uniqueness problems often encountered with plane-wave iterative phase retrieval. The method was tested on a cobalt/platinum multilayer exhibiting out of plane magnetized domains, where the magnetic circular dichroism effect was used to image the magnetic domains at the cobalt L3-edge at 780eV.

Strain-mediated ferromagnetism and low-field magnetic reversal in Co doped monolayer [Formula: see text].

Strain-mediated magnetism in 2D materials and dilute magnetic semiconductors hold multi-functional applications for future nano-electronics. Herein, First principles calculations are employed to study the influence of biaxial strain on the magnetic properties of Co-doped monolayer [Formula: see text]. The non-magnetic [Formula: see text] shows ferromagnetic signature upon Co doping due to spin polarization, which is further improved at low compressive (-2 %) and tensile (+2 %) strains. From the PDOS and spin density analysis, the opposite magnetic ordering is found to be favourable under the application of compressive and tensile strains. The double exchange interaction and p-d hybridization mechanisms make Co-doped [Formula: see text] a potential host for magnetism. More importantly, the competition between exchange and crystal field splittings, i.e. ([Formula: see text]), of the Co-atom play pivotal roles in deciding the values of the magnetic moments under applied strain. Micromagnetic simulation reveals, the ferromagnetic behavior calculated from DFT exhibits low-field magnetic reversal (190 Oe). Moreover, the spins of Co-doped [Formula: see text] are slightly tilted from the easy axis orientations showing slanted ferromagnetic hysteresis loop. The ferromagnetic nature of Co-doped [Formula: see text] suppresses beyond [Formula: see text] strain, which is reflected in terms of decrease in the coercivity in the micromagnetic simulation. The understanding of low-field magnetic reversal and spin orientations in Co-doped [Formula: see text] may pave the way for next-generation spintronics and straintronics applications.

Application of comet assay in the study of DNA damage and recovery in rohu (Labeo rohita) fingerlings after an exposure to phorate, an organophosphate pesticide.

Labeo rohita (rohu) fingerlings were exposed to different concentrations (0.001, 0.002 and 0.01 ppm) of phorate, an organophosphate pesticide; samplings were done at 24, 48, 72 and 96 h. The study was carried out to evaluate tissue specific genotoxic effects produced by phorate, on three different tissue systems and to assess DNA repair response in fish. Results of tissue specific DNA damage experiments showed low baseline damage in blood cells followed by gill and liver cells in control individuals whereas more DNA breaks were found in liver followed by gill and blood cells of treated individuals. Concentrations-dependent DNA damage showed a strong, linear and positive relationship (r(2) = >0.7) in all three tissues. Clear time-related increase in DNA damage was observed for all tissues exposed to all concentrations except in liver cells at 0.01 ppm, where the DNA damage declined significantly after 72 h. For the assessment of DNA repair response, fingerlings were first exposed to 0.01 ppm of phorate for 72 h and then transferred to pesticide free water. Tissue chosen for the repair experiment was liver. Samplings were done at 0, 3, 6, 12 and 24 h after the release of 72 h pesticide treated fishes into pesticide free water. Fishes showed a reduction in DNA breaks from 3 h onwards in pesticide free water and at 24 h returned to control level damage. The results indicate that phorate is a potential genotoxicant, comet assay can be used in DNA damage and repair analysis, response to pollutants in multicellular animals is often tissue specific.

Vaccination approach to prevent Argulus siamensis infection-success, challenges and preparedness.

•Argulosis, a disease caused by Argulus spp. of ectoparasites in scaly fish, is a global concern for aquaculture industry.•The resistance of the parasite to anti-parasitic drugs and the quantum of loss has been felt world-wide.•The current scenario of management and the development in vaccination are discussed herewith.

Contrasting solvent polarity effect on the photophysical properties of two newly synthesized aminostyryl dyes in the lower and in the higher solvent polarity regions.

Solvent polarity effect on the photophysical properties of two newly synthesized aminostyryl-thiazoloquinoxaline dyes, one with a flexible diphenylamino group, namely, N,N-diphenyl-4-[2-(thiazolo[4,5-b]quinoxalin-2-yl)vinyl]aniline (TQ1), and the other with a rigid julolidinylamino group, namely, (9-[2-(thiazolo[4,5-b]quinoxalin-2-yl)vinyl]julolidine) (TQ2), have been investigated in different aprotic solvents and solvent mixtures. From the polarity dependent changes in the absorption and fluorescence spectral properties, it is indicated that the fluorescent states of the dyes are of intramolecular charge transfer (ICT) character. For both the dyes, the photophysical properties like fluorescence quantum yields (Phi(f)), fluorescence lifetimes (tau(f)), radiative rate constants (k(f) = Phi(f)/tau(f)), and nonradiative rate constants (k(nr) = 1/tau(f) - Phi(f)/tau(f)) show clearly contrasting solvent polarity effects in the lower and in the higher solvent polarity region, causing an interesting reversal in the properties below and above an intermediate solvent polarity. It is inferred that the domination of the cis-trans isomerization in the lower solvent polarity region and that of the twisted intramolecular charge transfer (TICT) state formation in the higher solvent polarity region are responsible for the observed contrasting solvent polarity effects on the photophysical properties of the two dyes. As both isomerization and TICT state formation causes an enhancement in the nonradiative decay rate of the excited dyes and both the processes become less significant at the intermediate solvent polarity region, the two dyes show their largest Phi(f) and tau(f) values at intermediate solvent polarities. Suitable mechanistic schemes have been proposed and qualitative potential energy diagrams have been presented to explain the observed results with the changes in the polarity of the solvents used.

Magnetic Direct-Write Skyrmion Nanolithography.

Magnetic skyrmions are stable spin textures with quasi-particle behavior and attract significant interest in fundamental and applied physics. The metastability of magnetic skyrmions at zero magnetic field is particularly important to enable, for instance, a skyrmion racetrack memory. Here, the results of the nucleation of stable skyrmions and formation of ordered skyrmion lattices by magnetic force microscopy in (Pt/CoFeSiB/W)n multilayers, exploiting the additive effect of the interfacial Dzyaloshinskii-Moriya interaction, are presented. The appropriate conditions under which skyrmion lattices are confined with a dense two-dimensional liquid phase are identified. A crucial parameter to control the skyrmion lattice characteristics and the number of scans resulting in the complete formation of a skyrmion lattice is the distance between two adjacent scanning lines of a magnetic force microscopy probe. The creation of skyrmion patterns with complex geometry is demonstrated, and the physical mechanism of direct magnetic writing of skyrmions is comprehended by micromagnetic simulations. This study shows a potential of a direct-write (maskless) skyrmion (topological) nanolithography with sub-100 nm resolution, where each skyrmion acts as a pixel in the final topological image.

Effect of donor orientation on ultrafast intermolecular electron transfer in coumarin-amine systems.

Effect of donor amine orientation on nondiffusive ultrafast intermolecular electron transfer (ET) reactions in coumarin-amine systems has been investigated using femtosecond fluorescence upconversion measurements. Intermolecular ET from different aromatic and aliphatic amines used as donor solvents to the excited coumarin-151 (C151) acceptor occurs with ultrafast rates such that the shortest fluorescence lifetime component (tau(1)) is the measure of the fastest ET rate (tau(1)=tau(ET) (fast)=(k(ET) (fast))(-1)), assigned to the C151-amine contact pairs in which amine donors are properly oriented with respect to C151 to maximize the acceptor-donor electronic coupling (V(el)). It is interestingly observed that as the amine solvents are diluted by suitable diluents (either keeping solvent dielectric constant similar or with increasing dielectric constant), the tau(1) remains almost in the similar range as long as the amine dilution does not cross a certain critical limit, which in terms of the amine mole fraction (x(A)) is found to be approximately 0.4 for aromatic amines and approximately 0.8 for aliphatic amines. Beyond these dilutions in the two respective cases of the amine systems, the tau(1) values are seen to increase very sharply. The large difference in the critical x(A) values involving aromatic and aliphatic amine donors has been rationalized in terms of the largely different orientational restrictions for the ET reactions as imposed by the aliphatic (n-type) and aromatic (pi-type) nature of the amine donors [A. K. Satpati et al., J. Mol. Struct. 878, 84 (2008)]. Since the highest occupied molecular orbital (HOMO) of the n-type aliphatic amines is mostly centralized at the amino nitrogen, only some specific orientations of these amines with respect to the close-contact acceptor dye [also of pi-character; A. K. Satpati et al., J. Mol. Struct. 878, 84 (2008) and E. W. Castner et al., J. Phys. Chem. A 104, 2869 (2000)] can give suitable V(el) and thus ultrafast ET reaction. In contrary, the HOMO of the pi-type aromatic amines is largely distributed throughout the whole molecule and thus most of the orientations of these amines can give significant V(el) for ultrafast ET reactions with close-contact C151 dyes.

Host-guest complexation of neutral red with macrocyclic host molecules: contrasting pK(a) shifts and binding affinities for cucurbit[7]uril and beta-cyclodextrin.

The photophysical properties of the phenazine-based dye neutral red were investigated in aqueous solution in the presence of the macrocyclic host molecule cucurbit[7]uril (CB7) using ground-state absorption as well as steady-state and time-resolved fluorescence measurements. The results are contrasted to those previously obtained for beta-cyclodextrin (beta-CD; Singh et al. J. Phys. Chem. A 2004, 108, 1465). Both the neutral (NR) and cationic (NRH+) forms of the dye formed inclusion complexes with CB7, with the larger binding constant for the latter (K = 6.5 x 10(3) M(-1) versus 6.0 x 10(5) M(-1)). This result differed from that for beta-CD, where only the neutral form of the dye was reported to undergo sizable inclusion complex formation. From the difference in binding constants and the pK(a) value of protonated neutral red in the absence of CB7 (6.8), an increased pK(a) value of the dye when complexed by CB7 was projected (approximately 8.8). This shift differed again from the behavior of the dye with beta-CD, where a decreased pK(a) value (ca. 6.1) was reported. The photophysical properties of both NR and NRH+ forms showed significant changes in the presence of CB7. Fluorescence anisotropy studies indicated that the inclusion complexes of both forms of the dye rotate as a whole, giving rotational relaxation times much larger than that expected for the free dye in aqueous solution. The thermodynamic parameters for the NRH+.CB7 complex were investigated in temperature-dependent binding studies, suggesting an entropic driving force for complexation related to desolvation of the cation and the removal of high-energy water molecules from the CB7 cavity.

Hydrogen peroxide release from human eosinophils on fibronectin: scopoletin enhances eosinophil activation.

We have examined the release of H(2)O(2) from PAF or TNFalpha-stimulated human eosinophils on fibronectin (FN)-coated polystyrene plates. H(2)O(2) release was measured by the standard scopoletin-horseradish peroxidase (SCOP-HRP) method and compared with that measured by a new microplate fluorescent assay for H(2)O(2) using a novel HRP substrate A6550. We observed that the SCOP-HRP method gave a 25-fold higher estimate of H(2)O(2) release from eosinophils than did the A6550-HRP method. Microscopic examination of PAF or TNFalpha-stimulated eosinophils in buffer alone or A6550-HRP reaction mixture showed that the cells remained generally round, while eosinophils in SCOP-HRP reaction mixture were spread on the fibronectin-coated surface. Measurement of the cellular ATP content after PAF-stimulation showed that only eosinophils activated in SCOP-HRP had a 50% fall in ATP content. This supported our conclusion that measurement of H(2)O(2) release from eosinophils in SCOP-HRP reaction mixture is problematic since the SCOP-HRP system activates eosinophils. However, we also found that A6550-HRP, when present throughout the incubation, resulted in a lower estimate of H(2)O(2) release than expected. The method used to detect eosinophil H(2)O(2) release greatly influences the absolute amount of H(2)O(2) detected.

A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative.

This study describes a simple, reliable, highly sensitive and quantitative fluorescence microplate-assay of H2O2 from activated leukocytes using a novel horse radish peroxidase (HRP) substrate N-acetyl-3,7-dihydroxyphenoxazine (A6550). Unlike the widely used fluorescent HRP substrate scopoletin, A6550 is non-fluorescent and becomes highly fluorescent upon HRP-catalyzed H2O2 oxidation. Using 50 microM A6550, the change in fluorescence due to H2O2 generated from phorbol 12-myristate 13-acetate-activated human eosinophils and neutrophils is found to have a linear cell dose response up to 1.5 x 10(4) and 5 x 10(4) cells, respectively. The increase in fluorescence from A6550 is specifically due to H2O2 generation since it is inhibitable by catalase. Oxidized A6550 is found to be highly stable and the H2O2 dose response is linear as long as the ratio of A6550:H2O2 in the reaction mixture is higher than five. Unlike scopoletin, A6550 has a very low background, which changes little with time. In addition, the high fluorescent yield of oxidized A6550 results in an increased sensitivity for the detection of H2O2. When the concentrations of A6550 and HRP were 10 microM and 0.2 U/ml, respectively, as low as 2 pmol of H2O2 could be reliably measured. The sensitivity of A6550/H2O2 assay is found to be at least 10-fold higher than with scopoletin as the HRP substrate. The protocol described in this study using A6550 to measure H2O2 release from activated granulocytes can be easily adapted to other cell types which generate H2O2.

Attempts to purify a second cellular receptor for a coxsackievirus B3 variant, CB3-RD from HeLa cells.

A coxsackievirus B3 variant, CB3-RD, isolated on rhabdomyosarcoma (RD) cells is known to bind HeLa cells at two different receptor protein sites, HR1 and HR2. Since HR2 occurs in almost 50 fold excess of HR1 in HeLa cells, purification of HR2 was attempted, to obtain its partial N-terminal amino acid sequence and its further characterization. This study describes the purification of HR2 from octylthioglucoside solubilized HeLa cell membranes (HeLa-OTG) by preparative isoelectric focusing (IEF) followed by either preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or affinity chromatography on immobilized receptor monoclonal antibody, RmcA (RmcA-agarose). IEF of HeLA-OTG showed that both HR2 and HR1 could be well separated by this technique and focused with peak maxima around pH 3.7 and 6.7, respectively. Both RmcA and CB3-RD recognized HR2 as doublet bands (60 kD major polypeptide and a minor 55 kD polypeptide) on electroblots under non-reducing conditions. Preparative SDS-PAGE of the pool of IEF fractions containing HR2 (IEF pool) and simultaneous elution of polypeptides from the bottom of the gel during electrophoresis, is shown to be a useful technique in purifying HR2 with only one contaminating polypeptide (65 kD). However, affinity chromatography of the IEF pool on RmcA-agarose yielded HR2 without any detectable contaminating polypeptide. A quantitative chemiluminescence assay was developed to estimate the amount of HR2 on HeLa cells and in solution, when dot blotted on polyvinylidene difluoride (PVDF) membranes and probed with RmcA. Assays revealed that about 1.2% of the total HR2 present on HeLa cells could be obtained by IEF followed by affinity chromatography. Efforts are continuing to obtain sufficient quantities of purified HR2 for partial N-terminal amino acid sequencing.

Receptor proteins on newborn Balb/c mouse brain cells for coxsackievirus B3 are immunologically distinct from those on HeLa cells.

Newborn Balb/c mice are highly susceptible to infection by the six coxsackievirus serotypes of group B (CVB) and it is known that receptor for these viruses are in highest concentration in the brain as compared to other tissues. Therefore, proteins from the brain tissues of these animals were solubilized (Brain-Ext) and characterized for the identification of mouse brain receptor (MBR) proteins. Virus-blot analyses of Brain-Ext suggested that each of three virus variants of CVB3-(N, W and RD) recognized four receptor proteins designated p46, p44, p36 and p33 according to their molecular size. Similar analyses of cultured neurons from newborn Balb/c mice revealed the presence of the same four receptor proteins, while astrocytes appeared to possess only p46 and/or p44. Isoelectric focusing of Brain-Ext, focused MBR proteins in the pH range 4.0-8.5, with a peak around pH 5.7. P46 was found to be neuraminidase sensitive. A polyclonal rat antiserum (anti-MBR) protected cultured neurons and astrocytes against infection by CVB3, inhibited virus binding to these cells and recognized the same four receptor proteins on western-blots as detected on virus-blots by CVB3. However, a rabbit polyclonal anti-HeLa cell antiserum, which strongly binds to HeLa cells and protects them from CVB3 infection, neither recognized any of the receptor proteins in western-blot analyses of Brain-Ext nor inhibited CVB3 infection on cultured neurons and astrocytes. Conversely, anti-MBR did not recognize any of the receptor proteins by western-blot analysis of HeLa cell extracts nor did it inhibit CVB3 infection of HeLa cells.(ABSTRACT TRUNCATED AT 250 WORDS)