salicylates and 3-5-diiodosalicylic-acid

Topics: Cell Fractionation; Cell Nucleus; Chromatin; Intermediate Filaments; Iodobenzoates; Microscopy, Electron; Models, Biological; Nuclear Envelope; Nuclear Matrix; Nuclear Proteins; Salicylates; Sodium Chloride

Serum albumin (SA) is the most abundant protein in plasma and is the main transporter of molecules in the circulatory system of all vertebrates, with applications in medicine, the pharmaceutical industry and molecular biology. It is known that albumins from different organisms vary in sequence; thus, it is important to know the impact of the amino-acid sequence on the three-dimensional structure and ligand-binding properties. Here, crystal structures of ovine (OSA) and caprine (CSA) serum albumins, isolated from sheep and goat blood, are described, as well those of their complexes with 3,5-diiodosalicylic acid (DIS): OSA-DIS (2.20 Å resolution) and CSA-DIS (1.78 Å resolution). The ligand-free OSA structure was determined in the trigonal space group P3

Topics: Amino Acid Sequence; Animals; Binding Sites; Catalytic Domain; Cattle; Crystallization; Crystallography, X-Ray; Horses; Humans; Iodobenzoates; Models, Molecular; Protein Binding; Protein Conformation; Ruminants; Salicylates; Sequence Homology; Serum Albumin; Sheep

Due to their extraordinary binding properties, serum albumins are the main transporters of many small molecules in the circulatory system. Although all mammalian serum albumins exhibit quite high sequence similarity, their binding abilities are not the same. Until now, only human serum albumin (HSA) was subjected to extensive structural studies in complexes with various ligands. Here we present two crystal structures of the complexes of equine and bovine serum albumins with 3,5-diiodosalicylic acid (DIS), at resolutions 2.12 Å and 2.65 Å, respectively, and analyze interactions of the DIS ligand with both macromolecules. We highlight the differences in distribution of DIS binding sites between the bovine and equine serum albumins and compare results with the HSA binding ability of DIS and other structurally similar ligands.

Topics: Animals; Binding Sites; Cattle; Crystallography; Horses; Humans; Hydrogen Bonding; Iodobenzoates; Models, Molecular; Protein Binding; Protein Conformation; Salicylates; Serum Albumin

It has been shown that cell swelling stimulates the efflux of taurine from MCF-7 and MDA-MB-231 cells via a pathway which has channel-like properties. The purpose of this study was to examine the specificity of the volume-activated taurine efflux pathway in both cell lines. A hyposmotic shock increased the efflux of glycine, L-alanine, AIB (α-aminoisobutyric acid), D-aspartate but not L-leucine from MDA-MB-231 and MCF-7 cells. It was evident that the time course of activation/inactivation of those amino acids whose efflux was affected by cell swelling was similar to that of volume-activated taurine efflux. The effect of exogenous ATP on swelling-induced glycine, AIB and D-aspartate efflux from MDA-MB-231 cells was similar to that found on taurine efflux. In addition, volume-activated AIB efflux from MDA-MB-231 cells, like that of swelling-induced taurine efflux, was inhibited by diiodosalicylate. Tamoxifen inhibited volume-activated taurine release from both MDA-MB-231 and MCF-7 cells. The results suggest that neutral and anionic α-amino acids are able to utilize the volume-activated taurine efflux pathway in both cell lines. The effect of tamoxifen on breast cancer growth may, in part, be related to perturbations in cell volume regulation.

Topics: Adenosine Triphosphate; Alanine; Amino Acids; Aminoisobutyric Acids; Biological Transport; Breast Neoplasms; Cell Size; D-Aspartic Acid; Glycine; Humans; Iodobenzoates; Leucine; Osmolar Concentration; Salicylates; Tamoxifen; Taurine; Tumor Cells, Cultured

Rapid flow cytometric measurement of the frequency of aneuploid human sperms is in increasing demand but development of an exploitable method is hindered by difficulties of stoichiometric staining of sperm DNA. An aggressive decondensation protocol is needed after which cell integrity still remains intact. We used flow cytometry to examine the effect of lithium diiodosalicylate (LIS, chaotropic agent) on fluorescence intensity of propidium iodide-treated human spermatozoa from 10 normozoospermic men. When flow cytometric identification of diploid spermatozoa was achieved, validation was performed after sorting by three-color FISH. In contrast with the extremely variable histograms of nondecondensed sperms, consistent identification of haploid and diploid spermatozoa was possible if samples were decondensed with LIS prior to flow cytometry. A 76-fold enrichment of diploid sperms was observed in the sorted fractions by FISH. A significant correlation was found between the proportion of sorted cells and of diploid sperms by FISH. Application of LIS during the preparation of sperm for flow cytometry appears to ensure the stoichiometric staining of sperm DNA, making quantification of aneuploid sperm percentage possible. To our knowledge this is the first report in terms of separating spermatozoa with confirmedly abnormal chromosomal content. High correlation between the proportion of cells identified as having double DNA content by flow cytometry and diploid sperm by FISH allows rapid calculation of diploidy rate.

Topics: Diploidy; DNA; Flow Cytometry; Humans; In Situ Hybridization, Fluorescence; Iodobenzoates; Male; Propidium; Salicylates; Sensitivity and Specificity; Sperm Count; Spermatozoa

Transthyretin (TTR) is a plasma homotetrameric protein associated with senile systemic amyloidosis and familial amyloidotic polyneuropathy. In theses cases, TTR dissociation and misfolding induces the formation of amyloidogenic intermediates that assemble into toxic oligomeric species and lead to the formation of fibrils present in amyloid deposits. The four TTR monomers associate around a central hydrophobic channel where two thyroxine molecules can bind simultaneously. In each thyroxine binding site there are three pairs of symmetry related halogen binding pockets which can accommodate the four iodine substituents of thyroxine. A number of structurally diverse small molecules that bind to the TTR channel increasing the protein stability and thereafter inhibiting amyloid fibrillogenesis have been tested. In order to take advantage of the high propensity to interactions between iodine substituents and the TTR channel we have identified two iodinated derivatives of salicylic acid, 5-iodosalicylic acid and 3,5-diiodosalicylic acid, available commercially. We report in this paper the relative binding affinities of salicylic acid and the two iodinated derivatives and the crystal structure of TTR complexed with 3,5-diiodosalicylic acid, to elucidate the higher binding affinity of this compound towards TTR.

Topics: Crystallography, X-Ray; Halogenation; Humans; Iodine; Iodobenzoates; Prealbumin; Protein Conformation; Salicylates; Salicylic Acid

The purpose of this study was to determine the in-vitro induced nuclear chromatin decondensation (NCD) of human spermatozoa and its value in combination with routine semen analysis in predicting the outcome of in-vitro fertilization (IVF).. The ejaculate of 52 couples, undergoing IVF, was incubated with lithium diidosalicylic acid (LIS) and dithiothreitol (DTT) (G.1) or with heparin and sodium dodecyl sulphate (SDS) (G.2) to induce chromatin decondensation (NCD). Smears were made at 30, 60, and 120 min after incubation.. NCD was evaluated by morphometrical detection of the surface area of the spermatozoa using a semiautomatic image analysis system (IBAS). In both groups, the sperm heads showed a significant enlargement after 30, 60, and 120 min incubation in comparison to the initial size. However, no correlation was found between NCD at various periods of time and the fertilization rates. The mean area of the sperm heads in the native sample in the G.1 was 9.45+/- 1.33 microm(2) and 9.02+/- 1.15 microm(2) in the G.2. This area increased after incubation for 30, 60, and 120 min to 10.92 +/- 1.48, 12.26+/- 2.16, 13.54+/- 3.14, and 15.35+/- 7.78 microm(2) in the first group (G.1) and to 10.29 +/- 1.15, 11.23 +/- 1.85, 11.46 +/- 1.97, and 11.27 +/- 2.82 microm(2) in the second group, respectively.. NCD in vitro after incubation with LIS + DTT or heparin + SDS could not be recommended as a predictive parameter for IVF outcome.

Topics: Chromatin; Dithiothreitol; Female; Fertilization; Fertilization in Vitro; Heparin; Humans; In Vitro Techniques; Iodobenzoates; Male; Salicylates; Sodium Dodecyl Sulfate; Sperm Count; Spermatozoa

A large variety of DNA sequences have been described in nuclear matrix attachment regions. It could be most likely a result of the different methods used for their isolation. The idea about how different types of known DNA sequences (strongly attached to the nuclear matrix, weakly attached, or not attached) directly participate in anchoring DNA loops to the nuclear matrices isolated by different experimental procedures was tested in this study. Matrix-attached (M) and matrix-independent or loop (L) fractions as well as nuclear matrices were isolated using extractions of nuclei with 25 mM lithium 3,5-diiodosalicylate (LIS), 2 M NaCl, 0.65 M ammonium sulphate containing buffers followed by DNase I/RNase A digestion, or according to so designated conventional method. Using PCR-based and in vitro binding assays it was established that LIS and ammonium sulphate extractions gave similar results for the type of attachment of sequences investigated. The harsh extraction with 2 M NaCl or the conventional procedure led to some rearrangements in the attachment of DNA loops. As a result a big part of matrix attached sequences were found detached in the loop fractions. However, the in vitro binding abilities of the MARs to the nuclear matrices isolated by different methods did not change.

Topics: Ammonium Sulfate; Base Sequence; Binding Sites; Cell Fractionation; DNA; DNA Primers; HeLa Cells; Humans; In Vitro Techniques; Iodobenzoates; Nuclear Matrix; Polymerase Chain Reaction; Salicylates

Because the mechanisms that govern mitosis are a key to the understanding of cell growth, the proteins associated with chromosomes specifically during this phase have received thorough attention. In the present work we report an Mr58000 protein in MDCK epithelial cells, recognized by a monoclonal antibody (LFM-1) that decorates chromosomes during M-phase. Cell fractionation methods followed by immunoblotting and immunofluorescence showed that this protein is associated with the nuclear fraction. Biochemical extraction procedures on isolated metaphase chromosomes from nocodazole-synchronized cells indicated that the Mr58000 protein behaves as a chromosomal scaffold protein, that is, it remains in the pellets after high salt (2M NaCl) or 3'-5' diiodosalicylic acid treatments, even in DNAse pre-digested samples. In addition, confocal microscopy of those chromosomes revealed the LFM-1 epitopes distributed on the external surface and the axis of chromatids. Parallel analysis of interphase nuclei revealed LFM-1 epitopes inside G1-, but excluded from G2-phase nuclei. These results were independently confirmed on nuclei sorted by flow cytometry and in cell populations synchronized by release of G1-/S-phase hydroxyurea arrest. The Mr58000 and a minor Mr38000 protein (which was enriched only in mitotic chromosomes of synchronized cells) were analyzed by Edman degradation. They shared the sequence at the amino-terminal end but failed to show total homology with known proteins. These results suggest that LFM-1 antigens fit some of the predictions of the licensing factor model, and may have a role in cell cycle dependent events.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigens, Nuclear; Cell Cycle; Cell Line; Cell Nucleus; Chromatids; Dogs; Epithelial Cells; Epitopes; Interphase; Iodobenzoates; Mitosis; Molecular Sequence Data; Molecular Weight; Nocodazole; Nuclear Proteins; Salicylates; Sodium Chloride

It was shown that lipid composition of plant nuclear matrix depends on procedure of its isolation. The matrix isolated with the use of lithium diiodosalicylate (LiS) differs in its lipid composition from the preparation isolated with the use of nonionic detergent (Triton X-100). It was also shown that the nucleolytic activity of the matrix is related to its lipid component. Matrix depleted in lipids loses half of its nucleolytic activity which is recovered after supplementation with previously extracted lipids. The extent of recovery of the nucleolytic activity is also dependent on the presence of residual DNA in matrix preparation. The recoveries of nucleolytic activities were higher in matrices not depleted in their DNA content.

Topics: Iodobenzoates; Lipid Metabolism; Nuclear Matrix; Octoxynol; Plants, Edible; Salicylates

The properties of the conductive Cl- transport pathway underlying regulatory volume decrease (RVD) in human neutrophils were investigated using the whole cell patch-clamp technique. Cell swelling was induced during whole cell recordings by making the patch pipette solution hyperosmotic (approximately 20%) relative to the bath by addition of sucrose. Immediately after establishment of the whole cell configuration, no measurable Cl- currents were evident. Over a period of several minutes the outwardly rectifying Cl- current that developed displayed no apparent voltage dependence of activation and did not inactivate with time during voltage steps over the range of -80 to +80 mV. Reduction of Cl- currents by application of suction to the interior of the pipette implied that the swelling-induced Cl- channels are activated by membrane stretch. Based on reversal potential measurements, the volume-induced Cl- conductance was found to discriminate poorly among Cl-, Br-, I-, and NO3-, to possess a finite permeability to glucuronate (Pglucuronate/PCl approximately 0.1) and to be impermeable to cations. Single-channel conductance was estimated to be 1.5 pS from analysis of the variance of membrane current fluctuations. The activated Cl- currents were blocked by 100 microM of the compound MK-447 analogue A (inhibitor constant Ki = 37 microM) and by 200 microM 3,5-diiodosalicylate, 500 microM 4-acetamido-4'-iodothiocyanostilbene-2,2'-disulfonic acid, and 200 microM UK-5099. These results suggest that the initial event triggering RVD in neutrophils may be activation of stretch sensitive Cl- channels in the plasma membrane.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acrylates; Anions; Butylated Hydroxytoluene; Chlorides; Electric Conductivity; Humans; Iodobenzoates; Neutrophils; Salicylates; Water

Nuclear matrices were isolated from maize leaves by the two conventional methods usually employed for the preparation of the corresponding structures of animal origin. It is demonstrated that functionally competent matrices, recognizing and specifically binding the MAR-containing DNA of the mouse kappa-immunoglobulin gene may be prepared by both 2 M NaCl and LIS extractions of maize nuclei. A DNA region with a high affinity for the nuclear matrix was identified at the 5' end of the maize Adh1-S gene, distal to the promoter region. The presence of sites of reported altered chromatin structure in this particular region is discussed. While the proximity and the cohabitation of MARs with different regulatory elements is a common feature of matrix association regions in animal systems, this is the first plant MAR identified in a region of known significance for gene regulation.

Topics: Alcohol Dehydrogenase; Animals; Binding Sites; DNA; Immunoglobulin kappa-Chains; Iodobenzoates; Mice; Nuclear Matrix; Promoter Regions, Genetic; Restriction Mapping; Salicylates; Sodium Chloride; Zea mays

In immunoaffinity chromatography using monoclonal antibody, the elution conditions of an antigen, recombinant alpha-amylase, were studied. From among various conditions, three elution methods that gave fairly good yields of antigen activity (pH 2.3-2.5, pH 12.3-12.5 and 0.1 M lithium 3,5-diiodo salicylate [LIS]) were selected and the stabilities of the antigen and the antibody were analyzed. The antigen seemed to be eluted from the immunoadsorbent due to partial denaturation of either the antigen or the antibody. LIS seemed to be a specific denaturant for the antibody and its action was reversible. In terms of the stability of the antigen and repeated use of the immunoadsorbent, LIS seemed to be the best reagent for elution in immunoaffinity chromatography.

Topics: alpha-Amylases; Animals; Antibodies, Monoclonal; Antigens; Biotechnology; Chromatography, Affinity; Drug Stability; Hydrogen-Ion Concentration; Indicators and Reagents; Iodobenzoates; Mice; Protein Denaturation; Recombinant Proteins; Salicylates

1. The lithium diiodosalicylate/phenol method, widely employed for the isolation of membrane sialoglycoproteins (glycophorins) from mammalian erythrocytes, was applied for the first time to the purification of homologous glycoproteins from rat erythrocyte membranes. 2. The resulting preparations showed to be composed of four components, fractionated on SDS-PAGE. All four were positive for periodic acid-Schiff's reagent stain, the two largest of them being major. 3. Isolated rat glycophorins accounted for 60% of the ghost sialic acid and 1.5% of their protein. The presence of O-acetyl groups was confirmed in one-third of the sialic acid residues. 4. The molecular masses of the four glycophorin components were determined by a method which takes into account the anomalous mobility of glycoproteins on SDS-electrophoresis. Estimated values thus obtained for the actual molecular masses were 74, 32, 25 and 17 kDa.

Topics: Animals; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Glycophorins; Humans; Iodobenzoates; Lithium; Male; Rats; Rats, Wistar; Salicylates; Solubility

We have developed a new test to differentiate between ping-pong and simultaneous mechanisms for tightly coupled anion exchange. This test requires the use of a dead-end reversible noncompetitive inhibitor. As an example, we have applied the test to the anion exchanger of the HL60 cell using the salicylic acid derivative 3,5-diiodosalicylic acid (DIS), which reversibly inhibits HL60 cell Cl/Cl exchange. The concentration of DIS that causes 50% inhibition (ID50) increased only slightly as either intra- or extracellular chloride was increased, indicating that DIS inhibits HL60 anion exchange in a noncompetitive manner. In agreement with this observation, plots of the slope of the Dixon plot as a function of 1/[Clo] or 1/[Cli] were fit with straight lines with nonzero intercepts, indicating that DIS does not compete with either of the substrates ([Clo] and [Cli]). The secondary Dixon slope test is based on the fact that, for a dead-end inhibitor such as DIS, the slope of the Dixon plot slope vs. 1/[Cli] (secondary Dixon slope or SDS) is independent of extracellular Cl when the exchange mechanism follows ping-pong kinetics. Similarly, the SDS calculated from a plot as a function of 1/[Clo] is also independent of intracellular Cl for a ping-pong exchanger. In contrast to this prediction, we found that for DIS inhibition of Cl/Cl exchange in HL60 cells the slope of the Dixon plot slope vs. 1/[Cli] decreased by a factor of 2.5-fold when [Clo] was increased from 1 to 11 mM (P < 0.0001). This change in the SDS rules out ping-pong kinetics, but is consistent with a simultaneous model of Cl/Cl exchange in which there are extra- and intracellular anion binding sites, both of which must be occupied by suitable anions in order to allow simultaneous exchange of the ions.

Topics: Bicarbonates; Binding Sites; Chlorides; Extracellular Space; Humans; Intracellular Fluid; Iodobenzoates; Ion Transport; Kinetics; Models, Biological; Salicylates; Tumor Cells, Cultured

Different agents have been employed to extract the histones and other soluble components from isolated HeLa S3 nuclei during nuclear matrix isolation. We report that 0.2M (NH4)2SO4 is a milder extracting agent than NaCl and LIS (lithium 3,5-diiodosalicylate), on the basis of the apparent preservation of the elaborate fibrogranular network and the residual nucleolus that resemble the in situ structures in whole cells and nuclei, minimal aggregation, and sufficient solubilization of DNA and histones. The importance of intermolecular disulfide bonds, RNA and 37 degrees C stabilization on the structural integrity of the nuclear matrix was examined in detail using sulfydryl alkylating, reducing and oxidizing agents, and RNase A. The data suggest that any disulfides formed during the isolation are not essential for maintaining the structural integrity of the in vitro matrix. However, structural integrity of the matrix is dependent upon RNA and to some degree on disulfides that presumably existed in situ. Sodium tetrathionate and 37 degrees C stabilization of isolated nuclei resulted in nuclear matrices containing an approximately twofold greater amount of protein, RNA and DNA than control preparations. The 37 degrees C incubation, unlike the sodium tetrathionate stabilization, does not appear to induce intermolecular disulfide bond formation. Neither stabilizations resulted in significant differences of the major matrix polypeptide pattern on two-dimensional (2-D) gels stained with Coomassie Blue as compared to that of unstabilized matrix. The major nuclear matrix proteins, other than the lamins, did not react to the Pruss murine monoclonal antibody (IFA) that recognizes all known intermediate filament proteins, suggesting that the internal matrix proteins are not related to the lamins in intermediate filament-like quality.

Topics: Blotting, Western; Cell Fractionation; Cell Nucleus; Disulfides; DNA; Electrophoresis, Gel, Two-Dimensional; HeLa Cells; Histones; Humans; Hydrogen-Ion Concentration; Intermediate Filament Proteins; Iodobenzoates; Lamins; Microscopy, Electron; Nuclear Matrix; Nuclear Proteins; Nucleolus Organizer Region; RNA; Salicylates; Sodium Chloride; Solubility; Temperature; Tetrathionic Acid

Organotin derivatives represent a class of artificial ionophores that mediate Cl(-)-OH- exchange and thereby facilitate the chemical equilibrium distribution of Cl- and H+ across biological membranes. Imposing different pH and Cl- gradients by varying extracellular pH (pHo) and extracellular [Cl-] in the presence of 1 microM tributyltin validated the above assumptions in human neutrophils. Under relatively alkaline conditions [intracellular pH (pHi) greater than or equal to 7.10 and pHo greater than or equal to 7.40], the cell's natural Cl(-)HCO3- exchanger mimicked the actions of the tributyltin compound and was the principal factor controlling steady-state pHi. However, with increasing extracellular acidification, there was a progressive deviation from the predicted equilibrium distribution in the case of the normal Cl(-)-HCO3- transport system, whereas tributyltin-treated cells followed theoretical expectations. Exposure of neutrophils to a number of inhibitors of Cl(-)-HCO3- exchange led to a fall in pHi, apparently confirming the impression that a net HCO3- influx through Cl(-)-HCO3- countertransport was chiefly responsible for maintaining steady-state pHi. However, this intracellular acidification could be satisfactorily ascribed to proton movements through a parallel pathway, namely nonionic diffusion of the free acid form of the drugs. These results imply that Cl(-)-HCO3- exchange is the dominant pH regulatory device only under relatively alkaline conditions and that other mechanisms in addition to Na(+)-H+ exchange are likely to play an important role in recovery from acidification and in maintaining steady-state pHi. The possibility that the lactate carrier may function in this capacity is discussed.

Topics: Carrier Proteins; Chloride-Bicarbonate Antiporters; Chlorides; Dose-Response Relationship, Drug; Homeostasis; Humans; Hydrogen-Ion Concentration; Iodobenzoates; Kinetics; Mathematics; Neutrophils; Osmolar Concentration; Salicylates; Trialkyltin Compounds

Human sperm nuclei were isolated with mixed alkyltrimethylammonium bromide and dithiothreitol (MATAB/DTT) and decondensed by treatments with lithium diiodosalicylate (LIS), sodium chloride, or Tris salts. Concentrations as low as 1 mM LIS induced measurable nuclear swelling compared to 600 mM required for the other two salts. As measured by image analyses, the projected nuclear area increased linearly up to approximately fivefold with LIS concentrations up to 10 mM. Swollen nuclei also maintained the elliptical shapes characteristic of the human sperm head. Expanded sperm nuclei of three men were hybridized with a fluorescently labeled 3.4 kb Y chromosome-specific repetitive DNA probe; 50.1% of the nuclei of each semen sample showed fluorescent labeling over a part of the nucleus indicating presence of the Y chromosome. In comparison, unswollen sperm did not yield reliable hybridization signals. This procedure is suitable for determining the proportion of human sperm with Y chromosomes and can be used to evaluate sperm separation techniques. The availability of probes specific for most human chromosomes suggests that this procedure may find general application in studies of sperm chromosomal constitution.

Topics: Cell Nucleus; Chromatin; Dithiothreitol; DNA Probes; Fluorescence; Humans; Iodobenzoates; Lithium; Male; Quaternary Ammonium Compounds; Salicylates; Sodium Chloride; Spermatozoa; Y Chromosome

Chromaffin-granule membranes were separated into insoluble and soluble fractions after extraction with lithium di-iodosalicylate (LDIS). These fractions were characterized by one- and two-dimensional gel electrophoresis, and glycoproteins were detected after electroblotting with peroxidase-labelled concanavalin A and wheat-germ agglutinin (WGA). The LDIS-insoluble fraction contained components identified as glycoproteins III, H, J and K (carboxypeptidase H). Microsequence analysis indicated that component J is an N-terminally extended form of glycoprotein K. A major glycoprotein, GpII (Mr 80,000-100,000), present in the LDIS-soluble fraction was purified by affinity chromatography on WGA-Sepharose. This was characterized by one- and two-dimensional gel electrophoresis with Coomassie Blue staining, by amino acid analysis and automated N-terminal sequence analysis. Extraction of chromaffin-granule membranes with LDIS is a simple and rapid procedure that facilitates studies concerned with the structure and function of membrane glycoproteins from these and other secretory granules.

Topics: Adrenal Medulla; Amino Acid Sequence; Amino Acids; Animals; Cattle; Chromaffin Granules; Chromaffin System; Electrophoresis, Gel, Two-Dimensional; Iodobenzoates; Membrane Glycoproteins; Molecular Sequence Data; Receptors, Mitogen; Salicylates; Solubility

The effects of immunization in modulating the pathogenesis of Bacteroides (Porphyromonas) gingivalis infection in a murine model system were examined. BALB/c mice were immunized by intraperitoneal injection with B. gingivalis ATCC 53977 (one injection per week for 3 weeks), or with a lithium diiodosalicylate (LIS) extract (one injection per week for 3 weeks), or with lipopolysaccharide (LPS; one intravenous or intraperitoneal injection) from this same strain. Two weeks after the final immunization, the mice were challenged by subcutaneous injection of B. gingivalis ATCC 53977. Mice immunized with bacteria had no secondary lesions and no septicemia, whereas mice immunized with LIS extract had few secondary lesions and no septicemia. Mice immunized with LPS and nonimmunized mice demonstrated secondary abdominal lesions and septicemia after challenge. Bacterial cells and LIS extract, but not LPS, induced serum antibody and antigen reactive lymphocytes, as measured by enzyme-linked immunosorbent assay, immunoblot, Western immunoblot transfer, and in vitro lymphoproliferative responses. The present study suggests that immunization with a LIS extract or whole cells may induce a protective response against experimental B. gingivalis infection.

Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Outer Membrane Proteins; Bacteroides; Bacteroides Infections; Blotting, Western; Body Weight; Electrophoresis, Polyacrylamide Gel; Female; Immunization; Iodobenzoates; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Polysaccharides, Bacterial; Salicylates

3,5-Diiodosalicylate sodium (DIS), a highly lipophilic salicylate, was evaluated against 5-methoxysalicylate sodium (MS) as a potential adjuvant absorption promoter for rectal insulin delivery. Comparative blood glucose measurements were made using the two adjuvants under identical conditions as promoters of rectal insulin absorption in rats. Concentrations of DIS greater than and including 0.1 M produced an unexpected, progressive decrease in adjuvant activity as determined by a decline in observed hypoglycaemic response. This was not due to formation of an insulin-DIS complex. The adjuvant MS produced a classical, sigmoidal log-dose response curve. Possible reasons for the occurrence of the DIS optimum phenomenon are discussed as well as are the observed differences in adjuvant potency of these agents in a propylene glycol-containing vehicle.

Topics: Administration, Rectal; Animals; Biological Availability; Humans; Insulin; Intestinal Absorption; Iodobenzoates; Male; Rats; Rats, Inbred Strains; Rectum; Salicylates

Lithium diiodosalicylate (LIS) was used to selectively solubilize proteins from purified intestinal brush border membrane vesicles. Incubation of the vesicles with increasing concentrations of LIS resulted in the progressive release of proteins with total disruption of the membranes being obtained at 200 mM. Maximum selectivity was observed at 20-30 mM LIS which preferentially released actin and other non-glycosylated proteins while all the glycoproteins remained associated with the membrane. Electron micrographs showed that, after LIS treatment, brush border vesicles are partially disrupted and have lost their inner core of microfilaments. Sucrase, trehalase, leucylnaphthylamide hydrolase, gamma-glutamyl transpeptidase and alkaline phosphatase all retained more than 70% of their activities and remained associated with the membrane fraction after LIS solubilization (30 mM). The results indicate that lithium diiodosalicylate treatment provides an efficient method for the separation of cytoskeletal proteins from intrinsic membrane glycoproteins and should be very useful for the purification of microvilli proteins and for the study of membrane-protein interactions.

Topics: Animals; Electrophoresis, Polyacrylamide Gel; Intestinal Mucosa; Iodobenzoates; Male; Membrane Proteins; Microscopy, Electron; Microvilli; Rabbits; Salicylates; Solubility

Topics: Animals; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix; Female; Glycoproteins; Iodobenzoates; Male; Oocytes; Ovum; Salicylates; Sperm-Ovum Interactions; Swine; Zona Pellucida

Group A, type 12 streptococcal cell membranes were extracted by aqueous lithium diiodosalicylate (LIS) and the extract treated with trifluorotrichloroethane (Genetron). An initial component was isolated (GLCM) which was soluble in aqueous buffer, but was heavily contaminated with LIS. Dialysis of GLCM vs. 0.01 M Tris-EDTA (TE) buffer yielded a weakly antigenic component, TE-GLCM, with negligible contamination by LIS. Subsequent dialysis of TE-GLCM vs. isotonic calcium chloride produced two fractions, a soluble one (CMS) and a precipitate (CMP). It was demonstrated that CMS possessed immunological characteristics distinct from TE-GLCM and CMP. CMS was shown to be a calcium dependent antigen, and immunologically related to human glomerular basement membrane (GBM) antigens.

Topics: Amino Acids; Antigens, Bacterial; Basement Membrane; Calcium; Electrophoresis, Polyacrylamide Gel; Glomerulonephritis; Humans; Iodobenzoates; Kidney Glomerulus; Salicylates; Streptococcus pyogenes

Various methods of solubilization of human sperm antigens were compared by testing the sperm extracts against rabbit antihuman spermatozoa immune serums with double immunodiffusion and rocket immunoelectrophoresis. Marked differences were found in the protein contents and the SDS-PAGE protein patterns of the extracts. Lithium diiodosalicylate was the most efficient in solubilizing the sperm proteins and in preserving their antigenicity. Nonionic detergents also gave, either alone or combined to sonification and 1.0 M KCl, 24-28 clearly discernible protein bands and several different immunoprecipitates. Ionic detergents deoxycholate and cetylpyridinium chloride solubilized a high yield of proteins, but their antigenity was poor.

Topics: Animals; Antigens, Surface; Cetylpyridinium; Deoxycholic Acid; Detergents; Electrophoresis, Polyacrylamide Gel; Humans; Immunodiffusion; Immunoelectrophoresis; Iodobenzoates; Male; Mercaptoethanol; Octoxynol; Polyethylene Glycols; Rabbits; Salicylates; Solubility; Spermatozoa

Five glycoproteinmolecules with the molecular masses of 17 000; 38 000; 42 000; 50 000 and 67 000 were purified by high performance liquid chromatography following solubilization of isolated porcine zonae pellucidae by treatment with lithium-3,5-diiodosalicylate. The N-terminal amino acid residues were identified as arginine for 67 000, alanine for 50 000, arginine for 42 000, alanine for 38 000 and histidine for 17 000. The glycopeptides 42 000 and 17 000 were found to be rich on carbohydrates and 67 000 contained 7, 16% sialic acids. The latter moieties were tentatively identified as 5-N-acetylneuraminic acid, 5-N-glycolylneuraminic acid and 5-N-acetyl-7,8,9 tri-O-acetylneuraminic acid. The five components of the zona were resolved by thin layer chromatography in a solvent system of propanol/butanol/HCl (2:1:1) and showed Rf-values of 0.17, 0.42, 0.46, 0.50 and 0.55 respectively. The glycoprotein with the molecular mass of 38 000 possesses spermatozoal receptor properties. This receptor molecule showed a pI of 5.9 upon isoelectric focusing.

Topics: Amino Acids; Animals; Autoradiography; Carbohydrates; Chemical Phenomena; Chemistry; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Egg Proteins; Female; Glycoproteins; Iodobenzoates; Isoelectric Focusing; Molecular Weight; Ovum; Salicylates; Solubility; Swine; Zona Pellucida

The influence of treatments for extracting non-receptor peripheral proteins on the oligomeric states of the acetylcholine receptor has been studied in receptor-rich membranes from Torpedo marmorata. Conventional alkaline treatment of non-alkylated membranes resulted in the extraction of peripheral proteins (30% of total membrane proteins). Concomitantly, partial conversion of the dimer into the monomer was observed in the absence of exogenous reduction. Alkaline extraction at high ionic strength resulted in a marked decrease in protein solubilization, and no conversion of the dimer to the monomer occurred. Alkaline treatment extracted only one half of the peripheral proteins (15% of total protein) from membranes previously alkylated with N-ethylmaleimide or iodoacetamide, or oxidized by sodium periodate. Conversion of dimer to monomer was totally prevented by these treatments. Similar results were obtained by treatment of the membranes with lithium 3,5-diiodosalicylate. The above effects of alkaline extraction on the acetylcholine receptor can be interpreted in the context of two mutually non-exclusive mechanisms: (a) some of the peripheral proteins may directly participate in the thiol-dependent receptor aggregational states. Their extraction destroys this dynamic control. (b) Extraction of peripheral proteins destabilizes the receptor and makes it more susceptible to inter or intramolecular sulfhydryl-disulfide exchange, leading to the endogenous reduction of a proportion of the dimers.

Topics: Alkylation; Animals; Ethylmaleimide; Iodoacetamide; Iodobenzoates; Macromolecular Substances; Membrane Proteins; Microscopy, Electron; Osmolar Concentration; Periodic Acid; Receptors, Cholinergic; Salicylates; Torpedo

The isoantigenicity of rabbit germ cells and their extracts was studied by using a Western blot enzyme immunobinding procedure after systemic and local intrauterine immunization. In the deoxycholate (DOC) sperm extract, the antisperm isoantiserum (ASA) recognized proteins of 100, 96, 92, 82, 53, 47, 43, and 29 kD, and the antitestis isoantiserum (ATA) recognized proteins of 100, 96, 92, 82, 43, and 29 kD. In the lithium diiodosalicylate (LIS) sperm extract, ASA reacted with four antigen bands of 92, 30, 14, and 6 kD, and ATA reacted with proteins of 94, 92, 91, 23, 17, 7, and 6 kD. In the reduced sodium dodecyl sulfate (SDS) sperm extract, ASA recognized proteins of 95, 76, 51, 41, 33, 30, 28, 26, and 22 kD; ATA showed a reaction only with proteins of 100, 98, and 95 kD, and anti-LIS/sperm isoantiserum (ALA) recognized three proteins of 98, 81, and 65 kD. The IgA of the immune uterine fluid (IUF) from the LIS/sperm-immunized rabbits recognized a 14-kD antigen in the DOC conceptus extract, a 51-kD antigen in the DOC testis extract, and 53-, 49-, 31-, and 28-kD proteins in the reduced SDS-sperm pellet extract. The IgA of the IUF from the NP-40/pellet-immunized rabbits showed two proteins of about 100 kD, plus ones of 56, 36, 31, 29, 19, 17, and 14 kD in the DOC conceptus extract; proteins of 92, 70, 51, 29, and 14 kD in the DOC testis extract; and of 61, 57, 53, 49, 35, 31, and 28 kD in the reduced SDS-sperm pellet extract.

Topics: Animals; Deoxycholic Acid; Female; Immunoenzyme Techniques; Immunoglobulin A; Iodobenzoates; Isoantigens; Male; Octoxynol; Polyethylene Glycols; Rabbits; Salicylates; Sodium Dodecyl Sulfate; Spermatozoa; Testis; Uterus

Topics: Animals; Chromatography, High Pressure Liquid; Female; Glycoproteins; Iodobenzoates; Oocytes; Salicylates; Solvents; Swine

A specific system of transport for p-aminohippurate (PAH) is demonstrated in rabbit renal basal-lateral membrane vesicles. The PAH uptake into an intravesicular space is inhibited by probenecid in concentrations above 0.2 mM. The transport is saturable and is also temperature-dependent with an optimum between 37 and 45 degrees C. Divalent cations are able to enhance the uptake 2- to 3-fold. The stimulatory effect of the divalent cations diminishes in the following order: Mg++ = Mn++, Ba++, Ca++ and Sr++. Maximum stimulation occurs between 2.5 and 5 mM Mg++. The divalent cation stimulatory effect is not the result of changes in the size of the vesicles, in the degree of vesiculation, in the net charge of the membrane or of a transient potential difference across the membrane. Several inhibitors, more inhibitory than probenecid, were found. These are: lithium diiodosalicylate; 4-acetamido-4'-isothiocyano 2,2'-disulfonic acid stilbene; the mercurials, mersalyl acid, p-chloromercuriphenyl sulfonate and Hg++; and 5,5'-dithiobis(nitrobenzoate). Among these, mersalyl acid is the most potent inhibitor for PAH uptake. Its inhibitory effect is probably a combination of its reactivity toward sulfhydryl groups and its anionic character. The results with sulfhydryl reagents indicate that the PAH transport system contains sulfhydryl groups which are essential for the uptake activity. These sulfhydryl groups are probably buried in a hydrophobic region within the lipoprotein matrix of the basal-lateral membrane.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Aminohippuric Acids; Animals; Biological Transport; Cations, Divalent; Cell Membrane; Iodobenzoates; Kidney Cortex; Magnesium; Mersalyl; Organomercury Compounds; p-Aminohippuric Acid; Probenecid; Rabbits; Salicylates; Sulfhydryl Reagents

In guinea-pig Langendorff hearts, Na-salicylate (1.9, 3.8 and 7.6 mmol/l) concentration-dependently reduced the contractile force (--9.1, --51.0 and --75.1%, respectively) and the coronary resistance. The influence of the uncoupling agent 2.4-dinitrophenol (0.02 mmol/l) was comparable to that of the largest concentration of Na-salicylate. From the three congeners of Na-salicylate that were tested, only 3.5-diiodosalicylate (0.03 mmol/l) reduced the contractility whereas Na-benzoate (42 mmol/l) and m-iodobenzoate (1.2 mmol/l) were inactive. These results suggest that the cardiac effects of salicylate-like compounds in vitro are caused by an uncoupling of the oxidative phosphorylation.

Topics: 2,4-Dinitrophenol; Animals; Benzoates; Benzoic Acid; Depression, Chemical; Dinitrophenols; Guinea Pigs; Heart Rate; In Vitro Techniques; Iodobenzoates; Male; Myocardial Contraction; Oxidative Phosphorylation; Perfusion; Salicylates; Sodium Salicylate; Structure-Activity Relationship; Uncoupling Agents

In the series of salicylic acids derivatives investigated 3,5-DIS has a large inductive effect of enzymes with respect to the metabolism of xenobiotics: rise in the rate of Cyt. P 450; increase of ethoxycoumarin deethylase, bilirubin glucuronosyl transferase and benzphetamine-N-demethylase activities. This effect is comparable with that of phenobarbital.

Topics: 7-Alkoxycoumarin O-Dealkylase; Animals; Chemical Phenomena; Chemistry; Cytochrome P-450 Enzyme System; Enzyme Induction; Glucuronosyltransferase; Hexosyltransferases; Iodobenzoates; Male; Microsomes, Liver; Oxidoreductases, N-Demethylating; Oxygenases; Rats; Rats, Inbred Strains; Salicylates

Topics: Amino Acids; Avian Leukosis Virus; Avian Myeloblastosis Virus; Carbohydrates; Chromatography, Ion Exchange; Glycoproteins; Iodobenzoates; Lithium; Molecular Weight; Salicylates; Solubility; Viral Envelope Proteins; Viral Proteins

The lack of detailed studies of zona pellucida antigens up to the present time is partly due to the difficulty of the purification procedures involved. In the present work 4 porcine zona pellucida antigens were isolated using lithium 3,5-diiodosalicylate as the solubilizing agent, and immuno-affinity chromatography, with immobilized IgG, for purification. This method is believed to be more specific than the previous techniques used. The four zona antigens. ZPI/1 (Mr = 42000), ZPII/1 (Mr = 67000), ZPII/2 Mr=32000), and ZPIII/1 (Mr = 17000), were identified. Based on the reaction of the antigens with antibodies induced by intact and heat-solubilized zonae, it is postulated that only ZPI/1 and ZPII/1 are expressed on the surfaces of intact zonae. The cross reaction between human and porcine zona pellucida antigens is attributed to ZPI/1.

Topics: Animals; Antigens; Chromatography, Affinity; Female; Immunoglobulin G; Indicators and Reagents; Iodobenzoates; Lithium; Molecular Weight; Ovum; Salicylates; Solubility; Swine; Zona Pellucida

Glycophorin, the major sialoglycoprotein of the erythrocyte membrane, was extracted from human erythrocyte ghosts by the lithium diiodosalicylate phenol (LIS) or chloroform-methanol (CM) methods. The products (LISgp and CMgp) were examined for their capacity to inhibit invasion of erythrocytes by Plasmodium falciparum in vitro. In the presence of either glycoprotein, parasitemia was significantly less than in control cultures, indicating competitive inhibition of attachment. Desialylation resulted in only partial loss of this inhibitory potency. Neither crystalline NANA nor the dialyzates of either hydrolyzed glycoprotein had any inhibitory effect. We conclude that the receptor for merozoites of P. falciparum probably resides in the protein portion of glycophorin, in which NANA plays a secondary role, possibly related to hydration of the cell surface. The parasite itself contains no detectable neuraminidase activity.

Topics: Erythrocyte Membrane; Erythrocytes; Glycophorins; Glycoproteins; Humans; Iodobenzoates; Lithium; Plasmodium falciparum; Salicylates; Sialic Acids

Purified flagella from Euglena yield a unique high molecular weight glycoprotein when treated with low concentrations of nonionic detergents. This glycoprotein termed "xyloglycorien" cannot be extracted from other regions of the cell, although a minor component that coextracts with xyloglycorien does have a counterpart in deflagellated cell bodies. Xyloglycorien is tentatively identified with a flagellar surface fuzzy layer that appears in negatively stained membrane vesicles of untreated flagella but not in similar vesicles after Nonidet P-40 extraction. The localization of xyloglycorien is further confirmed to be membrane associated by reciprocal extraction experiments using 12.5 mM lithium diiodosalicylate (LIS), which does not appreciably extract xyloglycorien, visibly solubilize membranes, or remove the fuzzy layer. Rabbit antibodies directed against the two major flagellar glycoproteins (xyloglycorien and mastigonemes) to some extent cross react, which may in part be caused by the large percentage of xylose found by thin-layer chromatography (TLC) analysis to be characteristic of both antigens. However, adsorption of anti-xyloglycorien sera with intact mastigonemes produced antibodies responding only to xyloglycorien, and vice versa, indicating the nonidentity of the two antigens. Antibodies or fragments of these antibodies used in immunofluorescence assays demonstrated that xyloglycorien is confined to the flagellum and possibly the adjacent reservoir and gullet. Binding could not be detected on the cell surface. The sum of these experiments suggests that, in addition to mastigonemes, at least one major membrane glycoprotein in Euglena is restricted to the flagellar domain and is not inserted into the contiguous cell surface region.

Topics: Animals; Euglena gracilis; Flagella; Fluorescent Antibody Technique; Glycoproteins; Iodobenzoates; Lithium; Membrane Proteins; Molecular Weight; Salicylates; Solvents

Treatment of Newcastle disease virus with lithium diiodosalicylate differentially elutes the internally disposed proteins, M and NP, showing that these proteins are extrinsic, i.e., not associated with the lipid hydrophobic core. This selective elution requires disruption of the viral envelope, a process that is maximal at low temperature and influenced by the lipid composition of the virus envelope.

Topics: Electrophoresis, Polyacrylamide Gel; Iodobenzoates; Lithium; Newcastle disease virus; Salicylates; Temperature; Viral Proteins

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.

Topics: Alkalies; Animals; Cell Fractionation; Centrifugation, Density Gradient; Cholic Acids; Electrophoresis, Polyacrylamide Gel; Fishes; Iodobenzoates; Lithium; Microscopy, Electron; Receptors, Cholinergic; Receptors, Nicotinic; Salicylates; Synaptic Membranes

Topics: Animals; Cell Membrane; Cells, Cultured; Chemical Phenomena; Chemistry; Chromatography, Affinity; Collagen; Cricetinae; Cricetulus; Female; Fibronectins; Gelatin; Iodobenzoates; Lithium; Ovary; Salicylates; Sepharose

Topics: Cell Membrane; Electrophoresis, Polyacrylamide Gel; HeLa Cells; Humans; Iodobenzoates; Lectins; Lithium; Membrane Proteins; Molecular Weight; Neuraminidase; Phenols; Salicylates; Sialoglycoproteins; Trypsin

Glycoprotein was isolated from a purified thymocyte membrane preparation by two methods: lithium diiodosalicylate-phenol extraction and hot 75% ethanol extraction. A higher yield of membrane sialic acid was obtained by the latter method. The preparations had similar apparent molecular weights on sodium dodecyl sulfate gel electrophoresis. Both had similar receptor activities against a panel of hemagglutinins, although the 75% ethanol extract was more active on a weight basis. However, there were significant differences in carbohydrate and amino acid compositions of the two thymocyte extracts. The lithium diiodosalicylate-extracted material had much more glucose, ribose, and glycine than the ethanol extract. The glycoprotein preparations from thymocytes were quite distinct from the glycoprotein of bovine erythrocytes in both composition and receptor properties.

Topics: Animals; Biochemistry; Cattle; Cell Membrane; Cells, Cultured; Centrifugation, Density Gradient; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Ethanol; Glucose; Glycine; Glycoproteins; Hydrolysis; Iodobenzoates; Receptors, Mitogen; Ribose; Salicylates; Sialic Acids; Solvents; Sucrose; Thymus Gland; Time Factors

Topics: Animals; Cattle; Female; Iodides; Iodine; Iodine Isotopes; Iodobenzoates; Lactation; Potassium Iodide; Research; Salicylates; Thyroid Hormones; Thyroxine