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Biomedical subjects

M Wilchek

Publications and source records attributed to M Wilchek.

At least 127 records · Page 7Linked to original sources
PubMed1986

Selective covalent modification of newly synthesized activation-induced sialoglycoconjugates on the T lymphocyte membrane.

Resting human lymphocytes were oxidized by periodate and subsequently reduced with borohydride to block the aldehydes formed. The cells were then incubated for 24 or 48 hours with (or without) the mitogenic lectin phytohemagglutinin. A second oxidation with periodate at the indicated times resulted in strong surface-aldehyde formation in samples incubated with the mitogen, compared to control samples which exhibited very low quantities of aldehydes. The data show that this elevation in aldehyde formation was strictly dependent on protein synthesis, similarly to the appearance of Tac antigen in these cells. Cell surface aldehydes were detected in flow cytometry with a fluoresceinated hydrazide molecule and electrophoretically with biocytin hydrazide in conjunction with 125I-streptavidin. The proposed method for the elimination of the chemical reactivity of carbohydrates from the surface of resting lymphocytes thus enabled the selective covalent modification of newly synthesized sialoglycoconjugates formed upon a mitogenic trigger. The data also suggest the existence of a very low turnover rate of sialoglycoconjugates on the resting lymphocyte membrane.

Aldehydes↗
PubMed1986

Silica containing primary hydroxyl groups for high-performance affinity chromatography.

Primary hydroxyl groups were incorporated into silica by a four-step reaction procedure which includes modification of the silica surface with gamma-glycidoxypropyltrimethoxysilane, leading to an epoxide silica; hydrolysis with acid to yield a diol silica; oxidation of the diol silica with periodate to yield a silica resin with aldehyde functions; and reduction with sodium borohydride to obtain the primary hydroxyl-containing silica. The hydroxyl groups were activated with chloroformates or carbodiimidazole. Proteins were coupled under mild conditions in high yield to these activated silica resins. Columns containing these newly developed silica derivatives were used for the fast and efficient purification of antibodies on antigen-containing silica, as well as for the purification of trypsin on a trypsin inhibitor column (or vice-versa). The effect of pressure on association and dissociation of the affinity complex is discussed.

Antibodies↗
PubMed1986

A sensitive enzyme assay for biotin, avidin, and streptavidin.

Reciprocal enzyme assays are described for the vitamin biotin and for the biotin-binding proteins avidin and streptavidin. The assays are based on the following steps: (a) biotinylated bovine serum albumin is adsorbed onto microtiter plates; (b) streptavidin (or avidin) is bound to the biotin-coated plates; (c) biotinylated enzyme (in this case alkaline phosphatase) is then interacted with the free biotin-binding sites on the immobilized protein. For biotin assay, competition between the free vitamin and the biotinylated enzyme is carried out between steps (b) and (c). The method takes advantage of the four biotin-binding sites which characterize both avidin and streptavidin. The method is extremely versatile and accurate over a concentration range exceeding three orders of magnitude. The lower limits of detection are approximately 2 pg/ml (0.2 pg/sample) for biotin and less than 100 ng/ml (10 ng/sample) for either avidin or streptavidin.

Alkaline Phosphatase↗
PubMed1986

Lymphocyte activation by oxidative modification: avidin-modified T cells activate biotin-modified autologous T cells but not vice versa.

Immunocompetent human thymocytes were oxidized by periodate and conjugated with either biocytin-hydrazide or avidin-hydrazide followed by reduction with borohydride. Without further treatment, the resultant modified cells alone exhibited moderate levels of activation as a function of exogenously added interleukin 2. However, strong interleukin 2-dependent stimulation was observed when avidin-modified cells and biotin-modified cells were mixed. The effect was inhibited by free biotin molecules. By selective inhibition procedures, it was found that the avidin-modified cells induced the stimulation of biotin-modified cells, but not vice versa. It is postulated that the polyvalent avidin-hydrazide molecule covalently crosslinks and "freezes" the movement of oxidized glycoconjugates on the membrane surface. In contrast, the monovalent biocytin hydrazide would enable the modulation of membrane components upon interaction with avidin. The results suggest that crosslinking of mobile sites at the T-cell surface are essential for the transmission of an oxidation-induced mitogenic signal.

Avidin↗
PubMed1986

An improved method for the single-step purification of streptavidin.

A new method for the preparation of a more efficient, stable iminobiotin-containing resin for the isolation of streptavidin was developed. CL-Sepharose was activated with p-nitrophenyl chloroformate, and the resultant carbonate derivative was reacted with diaminohexane. Subsequent reaction of the amino-containing resin with iminobiotin-N-hydroxysuccinimide ester (in an organic solvent) yielded the stable affinity resin. The capacity of this resin for either avidin or streptavidin was 12 mg per ml resin, and streptavidin could be purified in one step directly from the culture broth of Streptomyces avidinii. The biotin-binding protein isolated in this manner exhibited a major band at about 75 kDa and a minor band at about 150 kDa. Under denaturing conditions, a spectrum of subunit molecular weights ranging between 15 and 19 kDa was detected, the distribution of which depended upon the specific preparation.

Bacterial Proteins↗
PubMed1986

Cis-platinum(II) complexes of carboxymethyl-dextran as potential antitumor agents. I. Preparation and characterization.

Cis-diamminedichloro platinum (II) (cis-DDP) and cis-diamminediaquo platinum (II) nitrate (cis-aq) were demonstrated to form complexes with dextran (dex) substituted with carboxymethyl (CM) groups at an average substitution ratio of 1 mole CM per 2 mole glucose units of dextran. The complexes were formed by reacting each of the two platinum (II) derivatives with carboxymethyl-dextran (CM-dex) at room temperature (RT) or at 37 degrees C in an aqueous solution. The complexing rate depended on temperature, ratio of platinum (II) compounds to CM-dex in the reaction mixture, and time of reaction. Experiments were performed with two CM-dex preparations, derived from dex T-10 (Mr-10,000) and from dex T-40 (Mr-40,000). Soluble cis-DDP and cis-aq complexes formed with CM-dex T-10 and CM-dex T-40 could carry up to 15 mole or 60 mole of the platinum (II) compounds per 1 mole CM-dex, respectively but higher complexing ratios resulted in complex precipitation. Reactivity of cis-aq with CM-dex was higher than that of cis-DDP. NaCl interfered with complex formation, but did not cause dissociation of already formed complexes. The binding of cis-DDP and cis-aq to CM-dex is, however, reversible since the drugs could be exchanged by other acceptors of higher affinity to platinum (II) such as O-phyenlenediamine, or DNA.

Antineoplastic Agents↗
PubMed1986

Cis-platinum (II) complexes of carboxymethyl-dextran as potential antitumor agents. II. In vitro and in vivo activity.

Cis-diamminedichloro platinum (II) (cis-DDP) and cis-diamminediaquo platinum (II) nitrate (cis-aq) were complexed to a macromolecular carrier carboxymethyl dextran (CM-dex). Two carriers were used in this study, one derived from dex-T-10 (Mr-10000) and the other from dex-T-40 (Mr-40000). The two platinum (II) drugs formed soluble complexes with both carriers. Uncomplexed and complexed drugs were tested and found to be cytotoxic in vitro against 5 murine and 2 human derived tumor cell lines. The two free platinum (II) drugs were cytotoxic against these cells to a similar extent. In comparison to the free drugs the complexes were somewhat less active, up to 3 fold, against murine 38C-13, L1210, EL-4 and RDM-4 leukemias, as well as against human HeLa and osteogenic sarcoma, and as active as the free drugs against murine F9 embryonal carcinoma. There were no major differences in the in vitro cytotoxic activity between CM-dex T-10 and CM-dex T-40 complexes. Differences due to the molecular size of the carrier were observed in vivo: The CM-dex T-10 complexes were significantly less toxic than the free drugs, whereas the reduction of toxicity by complexing to CM-dex T-40 was less profound. As for the efficacy, when tested in vivo against a cis-DDP sensitive tumor (F9) the T-40 complexes were equally or even more effective than the respective free drugs. The T-10 complexes were less effective than the free drugs at equal drug doses but their effectivity increased at increasing drug levels. These complexes were, however, very effective in inhibiting tumor growth upon repeated injections, leading to 100% survival.

Animals↗
PubMed1985

Blot analyses of glycoconjugates: enzyme-hydrazide--a novel reagent for the detection of aldehydes.

A procedure for the general staining of glycoproteins and other glycoconjugates on protein blots has been developed. Aldehydes are formed on the sugars of glycoconjugates by periodate oxidation which then react with hydrazide groups of enzyme-hydrazides, a novel reagent designed for aldehyde detection. The bound enzyme-hydrazide is demonstrated histochemically. The new assay is advantageous over periodic-acid Schiff staining of gels as its reagents and signals are stable and the process is simple and expedient, and provides greater sensitivity.

Aldehydes↗
PubMed1985

3-(N-Maleimido-propionyl)biocytin: a versatile thiol-specific biotinylating reagent.

A biotin-containing, thiol-specific reagent, 3-(N-maleimido-propionyl) biocytin (MPB), was synthesized and used to biotinylate various proteins via native or artificially induced sulfhydryl groups. In combination with appropriate avidin- or streptavidin-conjugated markers (i.e., fluorescent, enzyme-conjugated, electron-dense, etc.), MPB essentially constitutes a universal, multipurpose, thiol-specific probe. The reagent could be used to detect protein SH groups on dot blots with sensitivities in the femtomole range. The labeling was very specific for sulfhydryl groups or reduced S-S bonds; proteins lacking free SH groups were unlabeled by this method. Due to the long spacer between the biotinyl group and the reactive maleimide, improved adsorption of biotinylated proteins to avidin columns was achieved. An SH-containing enzyme (beta-galactosidase) was biotinylated with MPB, and the resultant biotinylated enzyme could be used as an efficient histochemical probe. The use of this reagent is recommended to biotinylate proteins which contain nonessential SH groups or which can be easily thiolylated prior to reaction with MPB.

Chemical Phenomena↗
PubMed1985

Cellular and growth factor requirements for the direct and indirect oxidative induction of interleukin 2 responsiveness in human thymocytes.

Human thymocytes were separated by peanut agglutinin (PNA) into PNA+ and PNA- cell fractions, and directly oxidized by the combined action of the enzymes neuraminidase and galactose oxidase (NAGO). Such treatment resulted in the induction of interleukin 2 (IL-2) responsiveness in PNA- cells but not in PNA+ cells. The molecular basis for the poor IL-2 responsiveness of PNA+ cells resides in their inability to express sufficient amounts of IL-2 receptors in response to NAGO treatment. Irradiated oxidized PNA+ or PNA- cells are able to transmit an oxidative mitogenic signal to autologous native PNA- cells but not to PNA+ cells. Depletion of plastic adherent cells from the PNA+ subpopulation totally abolished its high potency for the indirect signal transduction, whereas accessory cell depleted PNA- cells were affected to a lesser extent. Nonspecific esterase staining indicated that human thymic macrophages/monocytes are PNA+ cells. In spite of their small number (less than 0.5% of the total thymus cells) they appear to be very active in the indirect oxidative signal transmission. Unlike the indirect system, direct oxidative mitogenesis is independent of accessory cells. Attempts to detect NAGO-dependent IL-2 receptor inducing soluble factors were fruitless and there is a strict need for cell-cell interaction for the indirect transmission of the oxidative mitogenic signal.

Child, Preschool↗
PubMed1985

The covalent linking of two nucleotide analogues to antibodies.

Two anticancer drugs, antagonists of nucleic acids, were covalently linked to antibodies specifically reactive with B leukemia cells and thus with a potential possibility of drug targeting to the tumor site. The drugs cytosine 1-beta-D-arabinoside and 5-fluorouridine, competitive inhibitors of enzymes involved in DNA synthesis, were linked to antibodies via a dextran bridge. Cytosine 1-beta-D-arabinoside was linked to periodate-oxidized dextran and fluorouridine to dextran hydrazide. The dextran derivatives were in turn linked to antibodies that recognized a specific membrane IgM on B leukemia cells. The drug-antibody conjugates maintained most of the original antigen-binding capacity of the antibody and the full pharmacological activity of the drugs.

Animals↗
PubMed1984

Synthesis of pyridine derivatives of L-phenylalanine as antisickling reagents.

Several bicyclic agents composed of L-phenylalanine coupled to various pyridines were synthesized: 2-, 3-, and 4-(L- phenylalanylamino )pyridine. All three compounds at 3 mM gave positive morphological antisickling effects on homozygous SS cells under reduced O2 tension. Studies on two of these compounds, 2- and 3-(L- phenylalanylamino )pyridine, showed that these agents increase the deoxy-HbS solubility ratio, Cs/ Cs0 , by 14% at 20 mM. Observed changes in the mean corpuscular hemoglobin concentration (MCHC) values of treated cells ranged from 4% at 1.3 mM to 15% at 5.6 mM in compound concentration. Very minor lytic activity was found for treated cells, indicating water uptake is responsible for changes in the MCHC. Further, exposure of sickle cells to a 3 mM concentration of these agents also increased by 6- to 7-fold cellular deformability of a treated erythrocyte population as compared to an untreated one at the same total O2 saturation of 47%. These agents demonstrate the potential of bicyclic compounds composed of a common constituent, L-Phe, in the development toward a viable therapeutic agent.

Anemia, Sickle Cell↗
PubMed1984

Potential use of biaromatic L-phenylalanyl derivatives as therapeutic agents in the treatment of sickle cell disease.

N-Phenylacetyl-L-phenylalanine (PAP) and L-phenylalanyl-3-aminopyridine ( PAPA ) are biaromatic agents with properties that make them suitable candidates for the development of a useful therapeutic agent for the treatment of sickle cell disease. PAP and PAPA are taken up by the erythrocyte to give intra-/extracellular concentration ratios of 2.2 and 1.5, respectively, after a 2-hr exposure period. The intracellular buildup of PAP and PAPA produces moderate decreases in the mean corpuscular hemoglobin concentration (MCHC) of 6 and 10%, respectively, at 3 mM and a further decline in MCHC with increased concentration. Both PAP and PAPA increase the deoxy-Hb S solubility, CS. If the solubility in the absence of the agent is COS, PAP and PAPA have CS/COS values of 1.21 and 1.14 at 20 mM, respectively, compared with a value of 1.06 for L-phenylalanine itself. Filterability assays of partially dexygenated homozygous sickle cells shows an increase in cell flexibility of 7 to 16 times more than that of untreated cells when these agents are present at 3-6 mM. These results are largely due to the reduction in the Hb S polymer content of the treated cells. At 3 mM or less, both PAP and PAPA delay the onset of gelation in reversible sickle cells for time periods that are likely to be therapeutically useful.

Anemia, Sickle Cell↗
PubMed1984

Chemical reduction of oxidized human lymphocytes inhibits interleukin 2 production but not induction of interleukin 2 responsiveness.

Treatment with neuraminidase (NA) plus galactose oxidase (GalOxase) does not cause stimulation of human thymocytes. However, stimulation can be achieved by addition of exogenous interleukin 2 (IL-2). The IL-2-induced stimulation was inhibited with anti-Tac antibody, indicating that NA/GalOxase-oxidized cells can serve as inducers of functional IL-2 receptors on IL-2-responding T cells. The induction of IL-2 receptors by the oxidized cells was not inhibited by subsequent reduction with borohydride, since the cells could still be stimulated with IL-2. The presence of IL-2 receptors was also confirmed by flow cytometry using indirect immunofluorescence. Peripheral blood lymphocytes can be stimulated by NA/GalOxase treatment, and the conditioned medium from this treatment can support the growth of an IL-2-dependent line. This stimulation can be inhibited with borohydride and restored with IL-2. The conditioned medium derived from the borohydride-reduced cells cannot support the growth of the IL-2-dependent line, indicating that borohydride inhibits the oxidation-induced IL-2 production. The results suggest that NA/GalOxase-oxidized sites can be modified chemically without losing the potential to induce IL-2 receptors.

Cell Line↗
PubMed1984

Direct visualization of redistribution and capping of fluorescent gangliosides on lymphocytes.

Fluorescent derivatives of gangliosides were prepared by oxidizing the sialyl residues to aldehydes and reacting them with fluorescent hydrazides. When rhodaminyl gangliosides were incubated with lymphocytes, the cells incorporated them in a time- and temperature-dependent manner. Initially, the gangliosides were evenly distributed on the cell surface but were redistributed into patches and caps by antirhodamine antibodies. When the cells were then stained with a second antibody or protein A labeled with fluorescein, the fluorescein stain revealed the coincident movement of both the gangliosides and the antirhodamine antibodies. When the cells were treated with both rhodamine and Lucifer yellow CH-labeled gangliosides, the antirhodamine antibodies induced patching and capping of both fluorescent gangliosides but had no effect on cells incubated only with Lucifer yellow CH-labeled gangliosides. In addition, capping was observed on cells exposed to cholera toxin, antitoxin antibodies, and rhodamine-labeled protein A, indirectly showing the redistribution of endogenous ganglioside GM1, the cholera toxin receptor. By incorporating Lucifer yellow CH-labeled GM1 into the cells and inducing capping as above, we were able to demonstrate directly the coordinate redistribution of the fluorescent GM1 and the toxin. When the lymphocytes were stained first with Lucifer yellow CH-labeled exogenous ganglioside GM3, which is not a toxin receptor, there was co-capping of endogenous GM1 (rhodamine) and exogenous GM3 (Lucifer yellow CH). These results suggest that gangliosides may self-associate in the plasma membrane which may explain the basis for ganglioside redistribution and capping.

Animals↗