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Biomedical subjects

A A Branca

Publications and source records attributed to A A Branca.

16 recordsLinked to original sources
PubMed1995

Inhibition of the human immunodeficiency virus-1 protease and human immunodeficiency virus-1 replication by bathocuproine disulfonic acid Cu1+.

The protease encoded by the human immunodeficiency virus-1 (HIV-1) is essential for processing viral polyproteins which contain the enzymes and structural proteins required for the infectious virus. It was previously found that cupric chloride, in the presence of dithiothreitol or ascorbic acid, could inhibit the HIV-1 protease. It was suggested that a Cu1+ chelate was the moiety responsible for inhibition of the protease. This hypothesis has now been investigated directly by utilizing the stable Cu1+ chelate, bathocuproine disulfonic acid Cu1+ (BCDS-Cu1+). BCDS-Cu1+ inhibited the HIV-1 wild type protease as well as a mutant HIV-1 protease lacking cysteines. BCDS-Cu1+ was a competitive inhibitor of the mutant HIV-1 protease with an apparent Ki of 1 microM. Replication of HIV-1 in human lymphocytes and the cytotoxic effect of HIV-1 in CEM cells was inhibited by micromolar BCDS-Cu1+. Inhibition of the protease and of HIV replication by BCDS-Cu1+ was dependent on the presence of Cu1+ as BCDS alone was ineffective. EDTA blocked the inhibition of the protease by Cu1+ but was unable to block inhibition of the protease by BCDS-Cu1+, indicating that the Cu1+ complex was the inhibitory agent. The apparent IC50 for BCDS-Cu1+ on the inhibition of replication by primary isolates of HIV-1 was 5 microM. However, BCDS-Cu1+ did not affect polyprotein processing in an H9 cell line chronically infected with HIV-1, indicating that BCDS-Cu1+ acts by yet another mechanism to block HIV infection. Other possible targets for BCDS-Cu1+ include inhibition of viral adsorption and/or inhibition of the HIV-1 integrase.

Antiviral Agents↗
PubMed1989

Purification, measurement, and tissue distribution of a dansyl-derivatized glycopeptide from low-molecular weight follicle-stimulating hormone-inhibitor-containing fractions of porcine follicular fluid.

We have used dansyl chloride (5-dimethylamino-1-naphthalenesulfonyl choloride) to form dansyl derivatives of amine-containing compounds in follicular fluid or highly purified fractions containing a low molecular weight (MW) inhibitor of follicle-stimulating hormone (FSH) binding to receptor (FSH-BI). This approach allowed sensitive detection of the derivatives based on their fluorescent properties. By taking advantage of the hydrophobic nature of the dansyl group, a dansyl derivative (RF = 0.15) identified in low MW FSH-BI preparations was purified from porcine follicular fluid. Based on chromatographic criteria using four different systems (thin-layer chromatography [TLC] and high performance liquid chromatography), the derivatized factor (D15) that was purified appeared to be homogeneous. A direct, chemical assay was developed for quantification of D15 from follicular fluid or tissue extracts. The highest concentration (153 ng/mg) of D15 was found in ovarian tissue of adult rats, lesser amounts were observed in kidney and liver tissues (93 and 62 ng/mg, respectively) and even less in diaphram and heart tissues (5 and 0.5 ng/mg, respectively). High concentrations of D15 were observed in derivatized extracts of tests from immature rats in which approximately twice as much D15 was found in Leydig cells (241 ng/mg) as in seminiferous tubules (136 ng/mg). In porcine ovarian tissue, granulosa cells from large follicles and corpora lutea (69 and 91 ng/mg, respectively) contained at least 4-fold higher concentrations than follicle wall tissue (14 ng/ml). Relative concentrations of D15 material were also determined in pools of bovine follicular fluid previously shown to contain low MW FSH-BI.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Bovine spleen, a convenient source for purifying a type I interferon receptor.

Bovine spleen was investigated for the presence of receptors for radioiodinated human interferon-alpha 2 (125I-labeled HuIFN-alpha 2). Membranes were prepared by homogenization and differential centrifugation and analyzed for labeled IFN binding with recently developed tissue membrane assays. The characteristics of IFN binding included an affinity constant (Ka) of 3.1 +/- 0.99 X 10(10) M-1 and a receptor content of 8.4 +/- 0.74 fmoles/mg (wet weight) of bovine spleen membranes. The labeled IFN-receptor complex on these membranes was chemically cross-linked with 1.0 mM ethylene glycol bis(succinimidyl succinate (EGS), and subjected to SDS-PAGE and autoradiography. The formation of a 137-kD complex observed on autoradiographs was inhibited in a dose-dependent manner by unlabeled HuIFN-alpha 2 at concentrations that inhibited the binding of labeled IFN to the membranes. The products of the cross-linking reaction were purified by gel filtration on a column of Ultragel AcA34 in the presence of SDS and examined by SDS-PAGE and autoradiography. In addition to the 137-kD complex, several low-molecular-weight species were observed in the column profile of radioactivity which migrated to the bottom of 10% polyacrylamide gels. The fractions containing the 137-kD complex were pooled, concentrated, and utilized as a substrate for endoglycosidase digestion assays. Endoglycosidase H (EndoH) had no affect on the migration of the 137-kD complex while peptide:N-glycosidase F (PNGase F) increased the migration of the 137-kD band to a position with an Mr of 105 kD.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Quaternary structure of the calf testis follitropin receptor.

The quaternary structural relationships between subunits of the follitropin (FSH) receptor were determined through the use of the reversible, homobifunctional, chemical cross-linking reagent bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES) after formation of covalent 125I-azidobenzoyl-FSH-receptor subunit complexes by photoaffinity labeling. This experimental approach resulted in the formation of high molecular mass complexes (116, 172, and 320 kDa) as detected by autoradiography. After reversal of the BSOCOES cross-links, a second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of these complexes demonstrated that two lower molecular mass complexes, 64 and 84 kDa, identified previously as specific components of the FSH receptor by photoaffinity labeling alone (Smith, R. A., Branca, A. A., and Reichert, L. E., Jr. (1985) J. Biol. Chem. 260, 14297-14303) were contained within the 116- and 172-kDa complexes. In addition, the 116-kDa complex was not found to be a component of the 172-kDa complex. Since the high molecular weight complexes have molecular weights large enough to contain additional unlabeled proteins, these data indicate the possibility that there are several distinct FSH receptor subunits. Furthermore, the observation of the 320-kDa band, taken together with the observed structural relationships, suggests that the FSH receptor may contain two each of three distinct subunits with approximate molecular weights of 32,000, 48,000, and 86,000, respectively.

Affinity Labels↗
PubMed1986

High-affinity receptors for human interferon in bovine lung and human placenta.

Membrane fractions were prepared from fresh frozen bovine lung tissue and human placenta and used for receptor binding studies with recombinant DNA produced human-alpha-interferon (IFN-alpha). A single class of high-affinity binding sites was determined for bovine lung (Ka = 1.5 X 10(10) M) and human placenta (Ka = 3.7 X 10(9) M) membranes, respectively. These values for the affinity of IFN binding are comparable within the range of observed error to that observed for the binding of human IFN-alpha 2 to cultured Daudi cells (Ka = 2.5 X 10(10) M). The type I IFN receptor content of bovine lung and human placenta membranes was 1.3 and 2.5 fmoles/mg wet weight, respectively. In addition, alterations in specific and nonspecific binding were observed with the bovine lung membrane incubations in the presence of calcium. Increases in specific binding of three- to fourfold were observed in the presence of 1 mM calcium chloride.

Animals↗
PubMed1985

The subunit structure of the follitropin (FSH) receptor. Photoaffinity labeling of the membrane-bound receptor follitropin complex in situ.

Human follicle-stimulating hormone (hFSH) was acylated with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) and radioiodinated (55 microCi/micrograms) for use as a photoaffinity probe to investigate the subunit structure of the FSH receptor in calf testis. After incubation with the photoaffinity probe and photolysis with UV light, the cross-linked hormone-receptor complex was solubilized from the membrane and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Autoradiography of the polyacrylamide gels revealed two major bands, 64 kDa and 84 kDa. These were equivalent in molecular mass to those observed in a previous study (Branca, A. A., Sluss, P. M., Smith, A. A., and Reichert, L. E., Jr. (1985) J. Biol. Chem. 260, 9988-9993) in which performed hormone-receptor complexes were solubilized with detergent prior to formation of covalent cross-linkages through the use of homobifunctional cross-linking reagents. Reduction with dithiothreitol resulted in the loss of radioactivity from the 84-kDa band with a concomitant increase in the intensity of the 64-kDa band. Since dithiothreitol increases the dissociation of intact radioiodinated azidobenzoyl-FSH into subunits, it is suggested that the conversion of the 84-kDa band to the 64-kDa band by dithiothreitol is due to the loss of non-cross-linked hFSH subunit from the 84-kDa band and that the two bands observed after photoaffinity labeling arise from covalent bond formation between hFSH and a receptor subunit having a relative molecular weight (Mr) of 48,000. In addition to the predominant photolabeling of the receptor to yield the 64-kDa and 84-kDa bands, several other, less intense bands (54 kDa, 76 kDa, 97 kDa, and 116 kDa) were also consistently observed on autoradiographs. The appearance of all bands, however, was inhibited by the inclusion of unlabeled hFSH in the initial binding incubation mixtures. The results of this study indicate that the calf testis FSH receptor has a multimeric structure containing at least one 48-kDa subunit and suggest the presence of other nonidentical receptor subunit proteins.

Acylation↗
PubMed1985

The subunit structure of the follitropin receptor. Chemical cross-linking of the solubilized follitropin-receptor complex.

Homobifunctional cross-linkers were utilized to characterize high affinity (Ka = 2.2 X 10(-10) M-1) follitropin (FSH) receptors in immature bovine testis. Following the formation of radioiodinated human FSH (125I-hFSH)-receptor complexes, the membranes were solubilized with Triton X-100 or beta-octyl glucoside and the supernatants from ultracentrifugation (220,000 X g) subjected to gel filtration (Sephadex G-200) to separate the labeled hormone-receptor complexes from the unbound 125I-hFSH. The appearance of a high molecular weight (greater than or equal to 200,000) radioactive component in the elution profile was abolished when an excess of unlabeled hFSH was included in the initial incubation. After concentration by ultrafiltration, the 125I-hFSH-receptor complex, as well as the free hormone, was treated with a variety of chemical cross-linkers and subjected to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Bands of Mr = 65,000 and 83,000 observed in the autoradiograph of the hormone-receptor complex was not present in autoradiographs of free 125I-hFSH, nor were they present when an excess of unlabeled hFSH was included in the initial binding incubation mixtures. The 65,000 and 83,000 Mr bands were, therefore, considered to represent cross-linked complexes of labeled hFSH (Mr = 38,000) or its subunits (hFSH alpha, Mr = 16,000; hFSH beta, Mr = 21,000) and components of the FSH receptor. The bands were observed on autoradiographs when the extraction of the membranes was performed with either Triton X-100 or beta-octyl glucoside and when cross-linking was accomplished with disuccinimidyl suberate, ethylene glycol bis(succinimidyl succinate), or bis[2-(succinimido oxycarbonyl)oxyethyl]sulfone. The Mr of the native FSH receptor in the calf testis has been estimated at 146,000. Our studies demonstrate the multimeric nature of the FSH receptor. However, FSH is also composed of subunits, so that due to the complexity of the system, it was not possible to arrive at a precise assessment of the Mr or quaternary structure of the receptor subunits.

Animals↗
PubMed1983

Characterization of an interferon receptor on human lymphoblastoid cells.

A cell-free assay was developed to measure the binding of iodinated human interferon-alpha 2 to membranes prepared from lymphoblastoid Daudi cells. The kinetics of binding were similar at 0 degrees C and 30 degrees C, with 1.3-fold more interferon bound at the higher temperature. Membrane preparations treated with Triton X-100 proved to be a convenient source of solubilized receptor. An assay was developed to measure the binding of 125I-labeled interferon (125I-interferon) to solubilized receptors, based on the precipitation of interferon-receptor complexes with polyethylene glycol. Optimal binding with this assay was obtained at 0 degrees C. The solubilized receptor was analyzed by zonal sedimentation centrifugation and gel filtration. Sedimentation analysis in H2O and 2H2O gradients provided the sedimentation coefficient and the partial specific volume of the receptor-Triton X-100 complex. Gel filtration chromatography provided the Stokes radius of this complex. From these data we calculated several physical parameters, including Mr = 95,000 for the protein portion of the complex. The receptor is a highly asymmetric and hydrophobic membrane protein. 125I-Interferon could be crosslinked to receptors of intact Daudi cells or of isolated membranes by use of disuccinimidyl suberate. The covalently linked 125I-interferon-receptor complexes were analyzed by gel electrophoresis. A single band with Mr = 140,000 was detected in gel autoradiographs. If one molecule of interferon is present in this complex, the Mr of the receptor is close to 120,000. Possible reasons for the different Mr values obtained with the two analytical procedures used are discussed.

Burkitt Lymphoma↗
PubMed1983

Internalization and degradation of human alpha-A interferon bound to bovine MDBK cells: regulation of the decay and resynthesis of receptors.

The binding of 125I-labeled human interferon alpha-A (HuIFN-alpha A) to receptors of bovine MDBK cells was investigated. About 4-fold more 125I-interferon was bound at 37 degrees C than at 0 degrees C. To establish whether the cell-bound IFN was internalized, the cells were treated with diluted acetic acid, a procedure known to remove polypeptides bound to the cell surface. About 80% of the IFN bound at 0 degrees C was dissociated from the cells by this treatment, whereas only 45% of that bound after a 2 h incubation at 37 degrees C was dissociated. Release of cell-bound 125I-interferon by cells washed and incubated in fresh medium was next examined at the two temperatures. At 0 degrees C, up to 50% of cell-bound IFN was released into the medium over a 2 h period, whereas at 37 degrees C the cell-bound radioactivity was slowly released over several hours as acid-soluble degradation products. Interferon was therefore internalized and degraded by MDBK cells incubated at 37 degrees C, but not by cells incubated at 0 degrees C. The increased binding at 37 degrees C could possibly be explained by the internalization of IFN/receptor complexes and by the recycling of the receptors to the cell surface. This recycling was limited, however, since incubation of MDBK cells with unlabeled IFN led to a rapid decrease or down regulation of available receptors. Recovery of binding activity was prevented by the addition of inhibitors of protein and RNA synthesis, suggesting that de novo synthesis of receptors was required. The half-life of the IFN receptor in the presence of cycloheximide was about 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Down-regulation of the interferon receptor.

The binding of 125I-labeled alpha A interferon to human lymphoblastoid Daudi cells decreased when these cells were incubated with unlabeled alpha or beta interferon. This decrease could not be accounted for by the occupancy of interferon receptors with unlabeled interferon and it apparently resulted from the loss or down-regulation of receptors. The binding activity gradually increased when Daudi cells were incubated in fresh medium after a treatment with interferon, but inhibition of protein synthesis with cycloheximide prevented this recovery. Treatment of Daudi cells with this inhibitor resulted in the loss of half the interferon binding activity within 5 h. These findings suggested that the interferon receptors turn over at a basal rate in interferon-free medium and at an increased rate in cells incubated with interferon. The dose-response for the down-regulation was investigated by treating Daudi cells with different concentrations of alpha interferon. Down-regulation was observed in cells treated with relatively low doses of interferon, sufficient to elicit a biological response. The synthesis of the enzyme (2',5')oligo(A) polymerase was induced at the lowest interferon concentrations tested which caused receptor down-regulation.

Burkitt Lymphoma↗
PubMed1982

Interaction of interferon with cellular receptors. Internalization and degradation of cell-bound interferon.

Human interferon alpha A, produced in Escherichia coli by recombinant DNA technology, was labeled with 125I to study its binding to receptors on human lymphoblastoid Daudi cells. This binding showed a marked temperature dependency, with maximum binding obtained at 30-37 degrees C. About 60% of the cell-bound radioactivity was released upon subsequent addition of unlabeled interferon, indicating that only part of the cell-bound interferon could be displaced by competitor. Moreover, about 30-50% of cell-bound interferon was not released by treating the cells with 0.2 N acetic acid, a procedure which removes polypeptide hormones on the cell surface, indicating that part of the interferon bound at 37 degrees C was internalized. This interferon was slowly degraded to acid-soluble products, which were released into the culture medium. Treatment of DAudi cells with the lysosomotropic amines chloroquine and methylamine inhibited the degradation of interferon. Methylamine, however, also inhibited the internalization of interferon. Daudi cells treated with interferon in the presence of chloroquine showed an increase in the interferon-induced enzyme 2',5'-oligo(A) polymerase comparable to that of cells treated with interferon alone. This enzyme increased to a similar extent in cells treated with interferon and cytochalasin, a drug which inhibited internalization of interferon by 50%. These results suggest that degradation and possibly internalization of interferon are not required for at least some of its biological activities.

Biological Transport↗
PubMed1980

Inhibition of ornithine decarboxylase of HeLa cells by diamines and polyamines. Effect on cell proliferation.

1. Ornithine decarboxylase activity is stimulated in high-density HeLa-cell cultures by dilution of or replacement of spent culture medium with fresh medium containing 10% (v/v) horse serum. 2. After stimulation, ornithine decarboxylase activity reaches a peak at 4-6h, then rapidly declines to the low enzyme activity characteristic of quiescent cultures, where it remains during the remainder of the cell cycle. 3. The stimulation of ornithine decarboxylase is eliminated by the addition of 0.5mum-spermine or -spermidine or 10mum-putrescine to the HeLa-cell cultures at the time of re-feeding with fresh medium. Much higher concentrations (1mm) of the non-physiological diamines, 1,3-diamino-propane or 1,3-diamino-2-hydroxypropane, are required to eliminate the stimulation of ornithine decarboxylase in re-fed HeLa-cell cultures. 4. A heat-labile, non-diffusible inhibitor, comparable with the inhibitory protein ornithine decarboxylase antizyme, is induced in HeLa cells by the addition of exogenous diamines or polyamines. 5. Intracellular putrescine is eliminated, intracellular spermidine and spermine are severely decreased and proliferation of HeLa cells is inhibited when cultures are maintained for 48h in the presence of the non-physiological inducer of ornithine decarboxylase antizyme, 1,3-diamino-2-hydroxypropane. Exogenous putrescine, a physiological inducer of the antizyme, does not decrease intracellular polyamines or interfere with proliferation of HeLa cells.

Carboxy-Lyases↗