5'-Nucleotidase activity of mouse peritoneal macrophages. II. Cellular distribution and effects of endocytosis. (1/59)

The diazonium salt of sulfanilic acid (DASA) can inactivate about 80% of the total 5'-nucleotidase of viable macrophages. The remaining 20% can be inactivated if the cells are first lysed in detergent, and presumably represents an intracellular pool of 5'-nucleotidase. The bulk of this pool may represent cytoplasmic vesicles derived from plasma membrane by endocytosis. This internal compartment is expanded up to threefold immediately after the cells have ingested a large latex load. This is consistent with previous observations on the internalization of 5'-nucleotidase in latex phagosomes. In latex-filled cells this intracellular pool of enzyme is inactivated over a few hours, and the cells then slowly increase their enzyme activity to nearly normal levels. However, 24 h after latex ingestion the metabolism of 5'-nucleotidase in these recovered cells is abnormal, as the rate of enzyme degradation is about twice the normal rate, and the DASA-insensitive enzyme pool in these cells is strikingly diminished. This may reflect effects of the accumulated indigestible particles on the fate of incoming pinocytic vesicles or on newly synthesized plasma membrane precursor. Another endocytic stimulus, concanavalin A, also reduces the total cell 5'-nucleotidase activity. This effect, which is time and temperature dependent, can be prevented by the competitive sugar alpha-methyl mannose. The concanavalin A inhibition can be reversed in the absence of new protein synthesis or in cells cultivated in serum-free conditions. It is not known whether the effect of concanavalin A on 5'-nucleotidase depends upon the interiorizaiton of plasma membrane or is strictly associated with events at the cell surface.  (+info)

The oxygenase component of the 2-aminobenzenesulfonate dioxygenase system from Alcaligenes sp. strain O-1. (2/59)

Growth of Alcaligenes sp. strain O-1 with 2-aminobenzenesulfonate (ABS; orthanilate) as sole source of carbon and energy requires expression of the soluble, multicomponent 2-aminobenzenesulfonate 2,3-dioxygenase system (deaminating) (ABSDOS) which is plasmid-encoded. ABSDOS was separated by anion-exchange chromatography to yield a flavin-dependent reductase component and an iron-dependent oxygenase component. The oxygenase component was purified to about 98% homogeneity and an alpha2beta2 subunit structure was deduced from the molecular masses of 134,45 and 16 kDa for the native complex, and the alpha and beta subunits, respectively. Analysis of the amount of acid labile sulfur and total iron, and the UV spectrum of the purified oxygenase component indicated one [2Fe-2S] Rieske centre per alpha subunit. The inhibition of activity by the iron-specific chelator o-phenanthroline indicated the presence of an additional iron-binding site. Recovery of active protein required strictly anoxic conditions during all purification steps. The FAD-containing reductase could not be purified. ABSDOS oxygenated nine sulfonated compounds; no oxygen uptake was detected with carboxylated aromatic compounds or with aliphatic sulfonated compounds. Km values of 29, 18 and 108 microM and Vmax values of 140, 110 and 72 pkat for ABS, benzenesulfonate and 4-toluenesulfonate, respectively, were observed. The N-terminal amino acid sequences of the alpha- and beta-subunits of the oxygenase component allowed PCR primers to be deduced and the DNA sequence of the alpha-subunit was thereafter determined. Both redox centres were detected in the deduced amino acid sequence. Sequence data and biochemical properties of the enzyme system indicate a novel member of the class IB ring-hydroxylating dioxygenases.  (+info)

Mechanism of termination of immunological tolerance. (3/59)

Mice were rendered tolerant to bovine serum albumin (BSA) by means of cyclophosphamide. The effect of immunization with sulphanilated BSA with different degrees of hapten conjugation on this tolerant state was examined. It was shown that the tolerant state was readily terminated with sulphanilated BSA with a low degree of chemical modification. Specificities of antibodies produced in response to sulphanilated BSA were tested by absorption and PHA inhibition test, demonstrating that there were antibodies directed to new antigenic determinant(s) on sulphanilate BSA as well as those reacting with unmodified BSA. To explore the cellular mechanism of termination, lethally irradiated syngeneic mice were reconstituted with thymus-derived cells (T cells) together with bone marrow-derived cells (B cells) from either normal mice or mice rendered tolerant to BSA, and were immunized with BSA or sulphanilate BSA. Tolerance termination was clearly observed in the recipient mice reconstituted with tolerant T cells plus normal B cells. From these experimental data, it was proposed that the termination was effected by means of the co-operation of the T-cell population tolerant to BSA but capable of responding to new antigenic determinant(s) on sulphanilated BSA, with non-tolerant B cells responsive to BSA.  (+info)

Humoral and cell-mediated immunity in experimental progressive thyroiditis in rabbits. (4/59)

Rabbits immunized over a long period of time with serial injections of aqueous preparations of either bovine thyroglobulin or chemically altered rabbit thyroglobulin develop progressive thyroiditis. As is short-term thyroiditis in rabbits and mice, this thyroiditis is characterized by lesions and cellular infiltration similar to that observed in Arthus reactions. Once the progressive thyroiditis is established, the rabbits respond readily to subsequent injections of native rabbit thyroglobulin. No significant reduction of lesions or circulating antibody is observed when injections of native rabbit thyroglobulin are substituted for the preparations used to induce the disease. Cell-mediated hypersensitivity to rabbit thyroglobulin, as evidenced by MIF activity, develops in rabbits after prolonged immunization with altered or cross-reacting thyroglobulin. It is suggested that this activity develops as a result of a loss in the unresponsive state in T lymphocytes. The data indicate that it is the persistence of circulating antibody to autologous thyroglobulin which sequesters autologous thyroglobulin from peripheral lymphoid tissue, and thus, results in the loss of the unresponsive state in lymphocytes of these tissues. It is suggested that similar events may be involved in the development of cell-mediated hypersensitivity in thyroiditis in humans.  (+info)

Selenium spares ascorbate and alpha-tocopherol in cultured liver cell lines under oxidant stress. (5/59)

The selenoenzyme thioredoxin reductase (TR) can recycle ascorbic acid, which in turn can recycle alpha-tocopherol. Therefore, we evaluated the role of selenium in ascorbic acid recycling and in protection against oxidant-induced loss of alpha-tocopherol in cultured liver cells. Treatment of HepG2 or H4IIE cultured liver cells for 48 h with sodium selenite (0-116 nmol/l) tripled the activity of the selenoenzyme TR, measured as aurothioglucose-sensitive dehydroascorbic acid (DHA) reduction. However, selenium did not increase the ability of H4IIE cells to take up and reduce 2 mM DHA, despite a 25% increase in ascorbate-dependent ferricyanide reduction (which reflects cellular ascorbate recycling). Nonetheless, selenium supplements both spared ascorbate in overnight cultures of H4IIE cells, and prevented loss of cellular alpha-tocopherol in response to an oxidant stress induced by either ferricyanide or diazobenzene sulfonate. Whereas TR contributes little to ascorbate recycling in H4IIE cells, selenium spares ascorbate in culture and alpha-tocopherol in response to an oxidant stress.  (+info)

Regulation of insulin receptor function by a small molecule insulin receptor activator. (6/59)

In type 2 diabetes mellitus, impaired insulin signaling leads to hyperglycemia and other metabolic abnormalities. TLK19780, a non-peptide small molecule, is a new member of a novel class of anti-diabetic agents that function as activators of the insulin receptor (IR) beta-subunit tyrosine kinase. In HTC-IR cells, 20 microm TLK19780 enhanced maximal insulin-stimulated IR autophosphorylation 2-fold and increased insulin sensitivity 2-3-fold. In contrast, TLK19780 did not potentiate the action of insulin-like growth factor-1, indicating the selectivity of TLK19780 toward the IR. The predominant effect of TLK19780 was to increase the number of IR that underwent autophosphorylation. Kinetic studies indicated that TLK19780 acted very rapidly, with a maximal effect observed 2 min after addition to insulin-stimulated cells. In 3T3-L1 adipocytes, 5 microm TLK19780 enhanced insulin-stimulated glucose transport, increasing both the sensitivity and maximal responsiveness to insulin. These studies indicate that at low micromolar levels small IR activator molecules can enhance insulin action in various cultured cells and suggest that this effect is mediated by increasing the number of IR that are tyrosine-phosphorylated in response to insulin. These studies suggest that these types of molecules could be developed to treat type 2 diabetes and other clinical conditions associated with insulin resistance.  (+info)

New chromogen for assay of glucose in serum. (7/59)

We describe a colorimetric assay for glucose determination in human serum, with use of the chromogen 2-amino-4-hydroxybenzenesulfonic acid (AHBS), glucose oxidase, and peroxidase. With this assay, glucose concentrations less than or equal to 27.8 mmol/L can be measured in serum, with a sample/reagent volume ratio as low as 0.0025. The chromogen itself is easily soluble in water and does not require other components for the color change, making the reagent composition less complex. A single working reagent is used, and the reaction is completed within 10 min at 37 degrees C. The absorbance of the yellow reaction product is measured at 415 nm, and a blank sample measurement is not needed. The average analytical recovery of glucose in different human sera was 97.6%, with no significant interference of reducing compounds in serum. The results of the recommended procedure correlated well with those of the phenol/4-aminoantipyrine method of Trinder.  (+info)

Mineralization of sulfonated azo dyes and sulfanilic acid by Phanerochaete chrysosporium and Streptomyces chromofuscus. (8/59)

Five 14C-radiolabeled azo dyes and sulfanilic acid were synthesized and used to examine the relationship between dye substitution patterns and biodegradability (mineralization to CO2) by a white-rot fungus and an actinomycete. 4-Amino-[U-14C]benzenesulfonic acid and 4-(3-sulfo-4-aminophenylazo)-[U-14C]benzenesulfonic acid were used as representative compounds having sulfo groups or both sulfo and azo groups. Such compounds are not known to be present in the biosphere as natural products. The introduction of lignin-like fragments into the molecules of 4-amino-[U-14C]benzenesulfonic acid and 4-(3-sulfo-4-aminophenylazo)-[U-14C]benzenesulfonic acid by coupling reactions with guaiacol (2-methoxyphenol) resulted in the formation of the dyes 4-(3-methoxy-4-hydroxyphenylazo)-[U-14C]benzenesulfonic acid and 4-(2-sulfo-3'-methoxy-4'-hydroxy-azobenzene-4-azo)-[U-14C]benzenesulf oni c acid, respectively. The synthesis of acid azo dyes 4-(2-hydroxy-1-naphthylazo)-[U-14C]benzenesulfonic acid and 4-(4-hydroxy-1-naphthylazo)-[U-14C]benzenesulfonic acid also allowed the abilities of these microorganisms to mineralize these commercially important compounds to be evaluated. Phanerochaete chrysosporium mineralized all of the sulfonated azo dyes, and the substitution pattern did not significantly influence the susceptibility of the dyes to degradation. In contrast, Streptomyces chromofuscus was unable to mineralize aromatics with sulfo groups and both sulfo and azo groups. However, it mediated the mineralization of modified dyes containing lignin-like substitution patterns. This work showed that lignocellulolytic fungi and bacteria can be used for the biodegradation of anionic azo dyes, which thus far have been considered among the xenobiotic compounds most resistant to biodegradation.(ABSTRACT TRUNCATED AT 250 WORDS)  (+info)

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