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PubMed1976

Membrane perturbation: studies employing a calcium-sensitive dye, arsenazo III, in liposomes.

A metallochromic dye, arsenazo III [2,7-bis-(2-arsonophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid], has been incorporated into the aquenous interspaces of multilamellar liposomes. multilamellar liposomes. Addition of Ca produced no shift in the absorbance spectrum of dye captured by liposomes, whereas disruption of liposomes by Triton X-100, followed by Ca, produced the spectrum chracteristic of the dye-Ca complex: evidence of latency. Addition of excess ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) reversed the spectal shift. Differences between spectra obtained in this sequence yielded dye efflus. To measure Ca efflux, difference spectra (+/-EGTA) were obtained from cationic liposomes containing Ca after detergent lysis (sensitivity less than 10 mmol/ml). Since liposomes were impermeable either to dye or Ca until perturbed, it was possible to test a variety of membrane-active steroids (diethylstilbesterol, deoxycorticosterone, etiocholanolone) for their capacity to provoke dye efflux from liposomes; preincorporation of cortisol stablized liposomes against dye leak. Immunoglobulin-coated liposomes containing dye were taken up by phagocytes of Mustelus canis, and phagocytic vacuoles stained red-purple after ingestions. Liposomes containing the calcium-sensitive dye constitute a simple, accurate means for determining membrane perturbation and Ca fluxes; their uptake by cells or organelles remains to be exploited further.

Animals↗
PubMed1977

Membrane fusion: studies with a calcium-sensitive dye, arsenazo III, in liposomes.

Fusion between vesicles, cells, or organelles may be defined as confluence of two membrane-bound compartments without access of their solutes to external milieu. To study fusion by this criterion, we have trapped the metallochromic calcium-sensitive dye, arsenazo III (AIII), partially calcium-saturated (AIII-Ca) in one population of liposomes (phoshatidylcholine 90:dicetylphosphate 10), and ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetraacetate (EGTA) in a second. In such mixtures, interaction of EGTA with AIII-Ca was measured by a large color shift from blue leads to red (decreased absorbance at 660 nm). Fusion of liposomes (but also lysis and diffusion across the membranes) was proportional to these decrements. The exogenous "fusogens," lysolecithin and retinol, were added to liposomes for 5-24 hr at 37 degrees; after rechromatography, measurements were made of total dye, fraction of dye converted from AIII-Ca to AIII, and total lipid. After correction for lysis and diffusion, lysolecithin (200 microng/ml) induced 23% fusion (volume of AIII liposomes confluent with EGTA liposomes) and retinol (300 microng/ml) induced 15%. With one molar percent cortisol (a membrane stabilizer) in the liposome membranes, fusion induced by fusogens was reduced 2-fold. Neither multi-nor unilamellar liposomes fused with each other in the absence of exogenous fusogens, despite wide variations in molar lipid ratios. Results suggest that liposome-liposome fusion is a slow process requiring exogenous fusogens, which may depend upon contributions of other membrane constituents to mimic closely the fusion of natural membranes.

Arsenicals↗
PubMed1977

Resonance Raman spectroscopy of arsanilazocarboxypeptidase A: determination of the nature of the azotyrosyl-248-zinc complex.

Resonance Raman spectra of arsanilazotryosyl-248 carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.12.2) exhibit only the vibrational bands of its chromophoric azotyrosyl-248 residue uncomplicated by background interference from either water or other components of the protein. The resonance Raman spectra contain multiple, discrete bands which change as a function of pH, thereby demonstrating the existence of interconvertible species of the azotyrosine probe in solution. Spectra of model azophenols and of the apoazoenzyme establish the identity of these species. All conclusions about the azoenzyme based on the resonance Raman spectra, including the apparent pK values for the interconversion of these species, are in complete agreement with those drawn earlier from studies by absorption spectroscopy. In addition, the properties of resonance Raman bands that have been identified with the motions of specific atoms of azotyrosyl-248 provide details of the interactions of specific atoms of this chromophore with the catalytic zinc atom at the active site. In particular, this has allowed elucidation of the structure of the azotyrosyl-248-zinc coordination complex. Such experiments are also providing information on the effects of crystallization on the enzyme and on its interaction with inhibitors. The important potential of resonance Raman spectroscopy for the study of the structure of chromophoric components of active enzymatic sites and of metal complex ions is discussed.

Arsenicals↗
PubMed2005

Sorption kinetics of As(V) with iron-oxide-coated cement-a new adsorbent and its application in the removal of arsenic from real-life groundwater samples.

In the search of alternative adsorbents for the removal of arsenic, a new adsorbent (iron-oxide coated cement) was examined under various conditions to evaluate its suitability in removing As(V). A removal of more than 99% was obtained within 2 h for an initial As(V) concentration of 1 mg/L. Kinetic studies showed a removal which became somewhat constant after 2 h. Thorough investigations to understand the mechanism of solute adsorption onto the new sorbent with the help of four kinetic models, viz. approximately first-order reversible kinetics model, pseudo-first-order kinetic model, second-order kinetic model, and pseudo-second-order kinetic model showed that the sorption kinetics is consistent with the pseudo-second-order model from which it can be inferred that the mechanism of adsorption is chemical interaction or chemisorption. The equilibrium data followed the Langmuir isotherm at low concentrations of As(V) (<or=4 mg/L), but followed the Freundlich isotherm over a wide range of concentrations (0.5-10 mg/L) which can be due to solid solution precipitation. Studies on the effect of pH on As(V) removal revealed somewhat constant ( approximately 95%) As(V) removal over a wide pH range of 4-10. The efficiency of this adsorbent was also checked for the removal of arsenic from three real ground water samples containing both As(III) and As(V) with total arsenic in the range of 0.33-1.2 mg/L. The total arsenic removal was 71%, 87% and 75% for the samples containing 0.33, 0.88 and 1.2 mg/L total arsenic, respectively at the same adsorbent dose of 3 g/L. However, increasing the adsorbent dose will enhance removal capacity. Investigations of these real samples with the four kinetic models revealed most reliable fit with the pseudo-second order model. However, good fit was also obtained with the other three models. Values of the diffusion coefficients calculated from the first order kinetics data, indicated that, film diffusion is the rate limiting step for each of the three real life groundwater samples.

Adsorption↗
PubMed2007

Adsorption kinetics of natural dissolved organic matter and its impact on arsenic(V) leachability from arsenic-loaded ferrihydrite and Al-ferrihydrite.

The present work was broadly divided into two parts: first, the efficiency of synthetically prepared ferrihydrite and Al-ferrihydrite to adsorb dissolved organic matter (DOM) was tested as a function of time, pH and ionic strength and its effects on specific surface area was found. In the second part, the effect of DOM concentration and solution pH was studied to elucidate the influence of this two parameters on As(V) leachability from As(V) loaded ferrihydrite and Al-ferrihydrite. It was found that both pH and ionic strength had significant influence on DOM adsorption. The behavior and magnitude of adsorption was significantly different for both the materials. In general, the efficiency of Al-ferrihydrite to adsorb DOM was better compared to ferrihydrite. Results indicated that ligand exchange was the dominant interaction mechanism for DOM adsorption on ferrihydrite and Al-ferrihydrite. The study also suggested a strong potential for DOM to mobilize As(V) from the studied materials. At pH 5.0, DOM concentrations of 2.5 and 5.0 mg C/L had negligible effect on As(V) leachability. With increasing DOM concentration from 10 to 30 mg C/L, increased the As(V) leachability into the solution. Net release from ferrihydrite was up to 1.07 mg/g (5.75%) of the total adsorbed arsenic. The corresponding figure for Al-ferrihydrite was 1.47 mg/g (6.30%). As the primary mechanisms for the arsenic release from solid phases we identified two different types of mechanisms i.e., dissolution of the solid phase and competition between arsenic and organic anions for sorption sites.

Adsorption↗
PubMed2004

Separation of gallium and arsenic from the wafer grinding extraction solution.

This work investigates the separation of gallium and arsenic from the wafer grinding extraction solution. The wafer grinding extraction solution was generated using hot and concentrated nitric acid. In this study, adsorption technology was employed to remove the toxic arsenic from the extraction solution. Ferric hydroxide was the adsorbent employed to adsorb arsenic. The effects of pH value, contact time, absorbent dosage, and chloride ion concentration on the efficiency of adsorption of gallium and arsenic were investigated. The optimal conditions for recovering gallium and removing arsenic were a raw pH of 0.2, a contact time of 6min and a ferric hydroxide concentration of 30.4g/L. Additionally, adding chloric ions reduces the residual percentage of gallium (ReGa) and the percentage of arsenic removed (RAs). Under these optimal conditions, ReGa and RAs are 100 and 80%, respectively.

Adsorption↗
PubMed1966

Competition of haptens.

Groups of rabbits were injected with either bovine serum albumin, sheep red cell stroma, or keyhole limpet hemocyanin to which 2,4-dinitrophenyl and/or p-azophenyl arsonate groups had been coupled. Groups of animals received either doubly coupled antigen or an equivalent mixture of singly coupled antigens. Materials were injected intravenously as a solution or subcutaneously and intramuscularly in complete Freund's adjuvant. The presence of dinitrophenyl groups on the immunizing antigen could suppress, partially or completely, the antibody response to p-azophenyl arsonate when this hapten was located on the same molecule. Suppression was dependent on the ratio of haptenic groups on the molecule, appeared to be greatly affected by the method of immunization, and could be demonstrated in all three antigen systems. Partial suppression was manifested in decreased frequency and delayed appearance of the response as well as decreased maximal antibody titers. These findings appear irreconcilable with the possibility of direct clonal selection of antibody-producing cells by unprocessed antigen.

Animals↗
PubMed1967

Recovery of antibody activity from inactive hybrids of H and L chains.

Hybrid IgG molecules were prepared from the heavy and light chains of specifically purified antibody against two different haptens. One hybrid consisted of the H chains from the anti-p-azobenzoate antibody from one rabbit and L chains from the anti-p-azobenzenearsonate antibody from the second rabbit, and the second hybrid consisted of the opposite combination, light chains from the first rabbit and heavy chains from the second. The two hybrids were mixed at pH 8 and were found to be so stable in the mixture that even after 2 wk at 5 degrees C there was still only low hapten-binding activity toward p-iodobenzoate and p-iodobenzenearsonate. However, exposure of the mixture of hybrids to 1 M propionic acid followed by buffer at pH 8 resulted in recovery of binding activity for p-iodobenzoate and p-iodobenzenearsonate. Thus no exchange occurred between the light and heavy chains of the hybrids in the buffer, but exchange did occur on exposure to propionic acid, and this exchange favored a preferential combination among the chains in such a manner that effective antibody sites resulted.

Antibodies↗
PubMed1968

Studies on the competence of single cells to produce antibodies of two specificities.

Two series of experiments were performed, utilizing a modification of the hemolysin plaque technique which registers 19S antibody, in an attempt to determine the frequency of cells capable of simultaneously producing antibody to two non-cross-reacting antigens. Mice were immunized i.v. with rabbit and camel RBC and their spleens assayed for cells producing antibody against both antigens. 16,904 cells producing antibody of one or the other specificity, from 26 mice, were counted. Not one cell was detected which produced antibody of two specificities. Rabbits were immunized intradermally with HSA to which polyalanyl and p-azobenzenearsonate groups were chemically attached. The individual haptens, polyalanyl, and p-azobenzenearsonate groups were coupled to separate aliquots of SRBC, and the lymph nodes of immunized rabbits were assayed for cells releasing antibody against both haptens. In a study of 11 rabbits, after counting 27,845 cells producing antibody, we detected no "double" plaques.

Animals↗
PubMed1972

Suppression of idiotypic specificities in adult mice by administration of antiidiotypic antibody.

It has previously been shown that there are extensive idiotypic cross-reactions among antiphenylarsonate antibodies of A/J mice. The present work indicates that administration, into normal, adult A/J mice, of rabbit antiidiotypic antibody directed to A/J antiphenylarsonate antibody suppresses almost completely the subsequent production of antibody of the corresponding idiotype. No effect was noted on the formation of antibodies to the protein carrier or of antiphenylarsonate antibody of a different idiotype. The data are consistent with central suppression of production of the idiotypic antibody mediated through interaction with immunoglobulin receptors on lymphocytes.

Animals↗
PubMed1974

Hapten-sandwich labeling. I. A general procedure for simultaneous labeling of multiple cell surface antigens for fluorescence and electron microscopy.

A hapten-sandwich procedure has been developed for specific labeling of cell surface antigens for fluorescence or electron microscopy. Haptens are azo-coupled to immunoglobulins specific for a cell surface antigen; the hapten-modified cell-bound antibodies can then be visualized by adding fluorescent antihapten antibody, or by adding antihapten antibody followed by hapten-modified markers for electron microscopy. Virus or high molecular weight protein markers are lightly cross-linked before conjugation with hapten to prevent their disruption. Such stable hapten-modified markers, and the accessibility of many different purified anti-azophenyl-hapten antibodies, make it feasible to distinguish more than one membrane antigen in a given labeling experiment. When mouse lymphoid cell populations are labeled with separate markers for Ig and for thymus-associated antigens, many cells exhibit the Ig marker exclusively or the thymic marker predominantly, and some cells are completely free of label.

Animals↗
PubMed1975

Isolation from individual A/J mice of anti-rho-azophenylarsonate antibodies bearing a cross-reactive idiotype.

Immuization of A/J mice with a KLH-p-azophenylarsonate conjugate induces the formation of antihapten antibodies, some of which share idiotypic specificity common to all recipients. The subpopulation carrying the idiotype generally comprises 20-70% of the total antibody content. Large quantities of antihapten antibody (occasionally over 100 mg) were obtained from individual mice through the induction of an ascites fluid. This facilitated isolation of antibodies with the cross-reactive idiotype by isoelectric focusing. Most of this subpopulation has pI values between 6.65 and 6.95 and essentially all is of the IgG1 subclass. Two peaks, near pI 6.7 and 6.9, were frequently observed. Upon refocusing, the protein artifact of the procedure, but indicates microheterogeneity. The antibodies in the two peaks were found to be idiotypically identical by measurements of cross-inhibition. Preliminary studies have indicated that it is feasible to initiate investigations of primary structure with antibodies from individual inbred mice.

Animals↗
PubMed1975

Induction of T-lymphocyte responses to a small molecular weight antigen. II. specific tolerance induced in azebenzenearsonate (ABA)-specific T cells in Guniea pigs by administration of low doses of an ABA conjugate of chloroacetyl tyrosine in incomplete Freund's adjuvant.

The experiments presented in this paper demonstrate that the induction of tolerance on the one hand and the induction of delayed sensitivity on the other hand can be accomplished by administration of similar doses of azobenzene-arsonate conjugated to N-chloracetyl tyrosine (ABA-T) to guinea pigs with the determining factor being the absence or presence, respectively, of activating bacterial products in the adjuvant mixture used. Thus, complete, persistent ABA-T-specific T-cell tolerance can be induced in adult guinea pigs with 20 mug of ABA-T given intradermally in incomplete Freund's adjuvant (IFA) whereas this same dose of ABA-T induces ABA-specific immunity when administered in complete Freund's adjuvant. This tolerance was not reversible by administration of ABA-T and IFA in the presence of bacterial lipopolysaccharide, was generated before the formation of primed T cells, and persisted for at least 3 mo after initiation. Moreover, cell transfer studies performed herein demonstrate that the unresponsiveness resulting from administration of ABA-T in IFA reflects the activity of suppressor cells to induce and maintain a state of unresponsiveness could only be demonstrated in unprimed animals may indicate a severe limitation on the potential clinical usefulness of such an approach to regulation of the immune system.

Acetates↗
PubMed2004

Inorganic cadmium- and arsenite-induced malignant transformation of human bladder urothelial cells.

Arsenic and cadmium (Cd(+2)) are human carcinogens, and epidemiological studies have implicated both pollutants in the development of urinary bladder cancer. Despite this epidemiological base, it is unknown if either Cd(+2) or arsenite (As(+3)) can directly cause the malignant transformation of human urothelial cells. The goal of this study was to determine if Cd(+2) and/or As(+3) are able to cause the malignant transformation of human urothelial cells. The strategy employed was to expose the nontumorigenic urothelial cell line UROtsa to long-term in vitro exposure to Cd(+2) and As(+3), with the endpoint being the ability of the cells to form colonies in soft agar and tumors when heterotransplanted into nude mice. It was demonstrated that a long-term exposure to either 1 M Cd(+2) or 1 M As(+3) resulted in the selection of cells that were able to form colonies in soft agar and tumors when heterotransplanted into nude mice. The histology of the tumor heterotransplants produced by UROtsa cells malignantly transformed by Cd(+2) had epithelial features consistent with those of a classic transitional-cell carcinoma of the bladder. The histology of the tumor heterotransplants produced by cells malignantly transformed by As(+3) was unique in that the cells displayed a prominent squamoid differentiation.

Agar↗