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J H Shaper

Publications and source records attributed to J H Shaper.

At least 73 records · Page 4Linked to original sources
PubMed1985

Receptor function of mouse sperm surface galactosyltransferase during fertilization.

Past studies from this laboratory have suggested that mouse sperm binding to the egg zona pellucida is mediated by a sperm galactosyltransferase (GalTase), which recognizes and binds to terminal N-acetylglucosamine (GlcNAc) residues in the zona pellucida (Shur, B. D., and N. G. Hall, 1982, J. Cell Biol. 95:567-573; 95:574-579). We now present evidence that directly supports this mechanism for gamete binding. GalTase was purified to homogeneity by sequential affinity-chromatography on GlcNAc-agarose and alpha-lactalbumin-agarose columns. The purified enzyme produced a dose-dependent inhibition of sperm binding to the zona pellucida, relative to controls. To inhibit sperm/zona binding, GalTase had to retain its native conformation, since neither heat-inactivated nor Mn++-deficient GalTase inhibited sperm binding. GalTase inhibition of sperm/zona binding was not due to steric blocking of an adjacent sperm receptor on the zona, since GalTase could be released from the zona pellucida by forced galactosylation with UDPGal, and the resulting galactosylated zona was still incapable of binding sperm. In control experiments, when UDPGal was replaced with the inappropriate sugar nucleotide, UDPglucose, sperm binding to the zona pellucida remained normal after the adsorbed GalTase was washed away. The addition of UDPGal produced a dose-dependent inhibition of sperm/zona binding, and also dissociated preformed sperm/zona adhesions by catalyzing the release of the sperm GalTase from its GlcNAc substrate in the zona pellucida. Under identical conditions, UDP-glucose had no effect on sperm binding to the zona pellucida. The ability of UDPGal to dissociate sperm/zona adhesions was both time- and temperature-dependent. UDPGal produced nearly total inhibition of sperm/zona binding when the zonae pellucidae were first galactosylated to reduce the number of GalTase binding sites. Finally, monospecific anti-GalTase IgG and its Fab fragments produced a dose-dependent inhibition of sperm/zona binding and concomitantly blocked sperm GalTase catalytic activity. Preimmune IgG or anti-mouse brain IgG, which also binds to the sperm surface, had no effect. The sperm GalTase was localized by indirect immunofluorescence to a discrete plasma membrane domain on the dorsal surface of the anterior head overlying the intact acrosome. These results, along with earlier studies, show clearly that sperm GalTase serves as a principal gamete receptor during fertilization.

Acetylglucosaminidase↗
PubMed1985

Immunological localization and quantitation of the androgen-dependent secretory protease of the canine prostate.

We have recently reported on the isolation and characterization of the predominant protein of canine prostatic fluid and the subsequent identification of this protein as a proteolytic enzyme. In this report, we described the preparation and characterization of a rabbit antiserum against this secretory protease which was purified from canine prostatic fluid by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specificity of the antiserum has been established by immunoblot analysis and immunoprecipitation of the secretory protease present in canine prostatic fluid and tissue homogenates. The antiserum was used to localize (by immunofluorescence) and quantitate (by single radial immunodiffusion) the secretory protease in prostatic tissue obtained from intact and castrated dogs. These analyses can be summarized as follows: the secretory protease is strictly localized in the apical portions of the columnar epithelial cells in prostates of intact dogs, where it comprises almost 60% of the total Triton X-100-soluble protein; and the level of secretory protease decreases at least 500-fold in prostates of castrated dogs, demonstrating the androgen-dependent nature of this enzyme. Proteins immunologically related to the secretory protease were detected at low levels in the pancreas and salivary glands of the dog. Such proteins were not detectable in canine liver, kidney, vas deferens, testis, epididymis, lung, spleen, skin, small intestine, or sartorius muscle, suggesting a high degree of prostatic specificity for the secretory protease. These results demonstrate that the canine prostate is highly specialized with respect to its androgen-dependent synthesis and secretion of this secretory protease and that the antiserum specific for this protein can be used to assess canine prostatic function under varying hormonal conditions.

Animals↗
PubMed1984

Biological markers for breast carcinoma.

During the last decade, there has been an intense search for biological markers for breast carcinoma. Many different types of materials have been found that may be elevated in the body fluids of patients with this disease. However, no markers specific for breast cancer have been discovered and those currently available lack the sensitivity and specificity for early detection of the disease or for determining when the tumor burden is low. Problems may also occur in the interpretation of marker data due to apparent biological variations in synthesis or secretion. Plasma carcinoembryonic antigen (CEA), elevated in 60%-70% of patients with metastases, has had the most extensive evaluation. For the latter patients with increased plasma CEA, the levels in general are proportional to tumor burden. Changes in level with therapy correlate with measurable clinical parameters of response or progression in the majority of these patients. Specific patterns of serial CEA measurements after mastectomy may be helpful for predicting those patients most likely to develop recurrent disease. More recent attention has focused on trials of combinations of markers and on tissue measurement. The search for a specific marker for breast cancer using monoclonal antibody techniques is a promising area of considerable research interest.

Antibodies, Monoclonal↗
PubMed1984

A subset of non-histone nuclear proteins reversibly stabilized by the sulfhydryl cross-linking reagent tetrathionate. Polypeptides of the internal nuclear matrix.

When rat liver nuclei are isolated in the presence of the irreversible sulfhydryl-blocking reagent iodoacetamide, digested with DNase I and RNase A, and extracted with 1.6 M NaCl, nuclear envelope (NE) spheres depleted of intranuclear material, as analysed by thin-section electron microscopy, are obtained. Two-dimensional isoelectric focusing (IEF)/SDS-PAGE and non-equilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE reveal that the predominant polypeptides are lamins A, B and C. Nuclei isolated in the absence of sulfhydryl blocking reagents yield salt- and nuclease-resistant structures which contain sparse but demonstrable intranuclear material. A number of non-histone polypeptides are seen in addition to the lamins. Nuclei treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) yield, after exposure to nucleases and 1.6 M NaCl, nuclear matrix-like structures containing an extensive intranuclear network and components of the nucleolus in addition to the NE. Increased amounts of the non-lamin, non-histone polypeptides are recovered with these structures. Subsequent treatment of these NaTT-cross-linked structures with reducing agents in 1.0 M NaCl selectively solubilizes the intranuclear components but leaves the nuclear envelope apparently intact. The lamins remain sedimentable and are virtually absent from the soluble (intranuclear) material. Instead, the major solubilized polypeptides are (a) 68 and 63 kD polypeptides which migrate in the vicinity of lamins B and C, respectively, but are distinguishable from the lamins by immunoblotting and by uni-dimensional peptide mapping; (b) a series of basic 60-70 kD polypeptides (pI greater than 8.0) which are not recognized by anti-lamin antisera; (c) an acidic (pI 5.3) 38 kD polypeptide; and (d) a number of high molecular mass (greater than 100 kD) polypeptides. These observations not only suggest a convenient method for fractionating matrix structures from rat liver nuclei into biochemically and morphologically discrete components, but also identify a subset of major non-lamin, non-histone nuclear polypeptides (comprising approx. 20% of the total nuclear protein) whose intermolecular interactions can be reversibly stabilized apparently by intermolecular disulfide bond formation by NaTT.

Animals↗
PubMed1984

Binding of dexamethasone to rat liver nuclei in vivo and in vitro: evidence for two distinct binding sites.

The binding of [3H]dexamethasone (DEX) to rat liver nuclei in vitro and in vivo have been compared. In vitro, purified nuclei displayed a single class of specific glucocorticoid binding sites with a dissociation constant (Kd) of approximately 10(-7) M for [3H]DEX at 4 degrees C. The glucocorticoid agonists prednisolone, cortisol, and corticosterone and the antagonists progesterone and cortexolone competed avidly for this site, but the potent glucocorticoid triamcinolone acetonide (TA) competed poorly in vitro. Nuclei isolated from the livers of intact rats contained 1-2 X 10(4) [3H]DEX binding sites/nucleus. Up to 85% of the binding sites were recovered in the nuclear envelope (NE) fraction when NE were prepared either before or after labeling with [3H]DEX in vitro. After adrenalectomy, the specific [3H]DEX binding capacity of both nuclei and NE decreased to 15-20% of control values, indicating sensitivity of the binding sites to hormonal status of the animals. Efforts to restore the binding capacity by administration of exogenous glucocorticoids, however, were unsuccessful. After labeling of rat liver nuclei in vivo by intraperitoneal injection of [3H]DEX or [3H]TA into living animals, the steroid specificity and subnuclear localization of radiolabel were different. Both [3H]TA (which did not bind in vitro) and [3H]DEX became localized to nuclei in a saturable fashion in vivo. With either of these ligands, approximately 20% of the total nuclear radiolabel was recovered in the NE fraction. These results suggest the presence of two separate and distinct binding sites in rat liver nuclei, one which is localized to the NE and binds [3H]DEX (but not [3H]TA) in vitro, and another which is not localized to the NE but binds [3H]DEX and [3H]TA in vivo.

Animals↗
PubMed1984

Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells.

The anti-My-10 mouse monoclonal antibody was raised against the immature human myeloid cell line KG-1a and was selected for nonreactivity with mature human granulocytes. Anti-My-10 immunoprecipitated a KG-1a cell surface protein with an apparent Mr of approximately 115 kD. We describe the binding of this antibody to human hematopoietic cell types and show that My-10 is expressed specifically on immature normal human marrow cells, including hematopoietic progenitor cells. My-10 is also expressed by leukemic marrow cells from a subpopulation of patients. Thus, this antibody allows the identification and purification of hematopoietic progenitor cells from normal human marrow and the subclassification of leukemia.

Acute Disease↗
PubMed1983

Isolation and characterization of the major androgen-dependent glycoprotein of canine prostatic fluid.

Canine prostatic fluid, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, is characterized by the presence of a single diffuse band (Mr approximately 30,000) which accounts for over 90% of the total protein. The biosynthesis of this protein is under androgen control. Castration results in the disappearance of this protein, whereas its presence in the prostate can be maintained in the castrated animal with exogenous androgen. Analysis of the native protein by isoelectric focusing revealed the presence of 10-13 charged variants with pI values in the range of 6.5 to 8.4. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed that each isoform is constructed of two dissimilar polypeptide subunits covalently linked through disulfide bonds. One subunit has a molecular weight of 15,000 (H chain); the second subunit (L chain) has a variable molecular weight in the 12,000-14,000 range. The H and L subunits have been purified by preparative isoelectric focusing and chemically characterized. Based on tryptic peptide mapping, NH2-terminal analysis, amino acid and carbohydrate composition, the H and L subunits are structurally unrelated and consequently appear to be unique gene products. Furthermore, the L subunit is glycosylated which potentially accounts for its size heterogeneity. Quantitative NH2-terminal analysis indicated that the H and L subunits are present in the native molecule in a ratio of 2:1, suggesting that the native molecule is a trimer with an apparent molecular weight of 43,000. Based on electrophoretic data, the glycoprotein also constitutes the major fraction of the soluble protein in canine prostatic tissue; its presence is organ specific. This glycoprotein should prove useful as a marker for prostatic function under varying hormonal and environmental conditions.

Amino Acids↗
PubMed1983

Analysis of cell surface proteins delineates a differentiation pathway linking endocrine and nonendocrine human lung cancers.

We have previously determined that the cell surface protein phenotype distinguishes human small cell lung carcinoma (SCC), a neoplasm with endocrine properties, from non-SCC in culture. We now demonstrate that cloned cell cultures of human large cell undifferentiated lung carcinoma, established directly from a patient with mixed SCC and non-SCC, simultaneously express surface proteins characteristic of SCC and non-SCC lung cancer cells. Hence, SCC and a form of large cell carcinoma appear linked through a continuum of differentiation events that: (i) may explain clinically important transitions that occur between the major types of human lung cancer and (ii) provide evidence for a common cellular origin of endocrine and nonendocrine cells in the bronchial mucosa.

Amine Oxidase (Copper-Containing)↗
PubMed1983

Tunicamycin inhibits ganglioside biosynthesis in neuronal cells.

The antibiotic tunicamycin blocks the transfer of GlcNAc-1-P from UDP-GlcNAc to dolichol phosphate, thereby blocking the synthesis of N-linked oligosaccharide chains on glycoproteins. Its effect on the biosynthesis of gangliosides has not been reported. We report that tunicamycin caused a 70-80% reduction in incorporation of [(3)H]GlcN into gangliosides and neutral glycosphingolipids of the neuroblastoma-glioma hybrid cell line NG 108-15 at antibiotic concentrations that caused a 90% reduction of the radiolabel incorporation into glycoproteins. The effect of tunicamycin on ganglioside biosynthesis was apparent after only 4 hr of incubation, and maximum inhibition was seen within 6 hr. When control or tunicamycin-treated (5 mug/ml) cells were collected and fractionated to separate glycoproteins, neutral glycosphingolipids, gangliosides, and nucleotide sugar-precursor pools, the following results were obtained: (i) UDP-GlcNAc and UDP-GalNAc pool sizes increased >3-fold, and specific activities decreased 50% upon treatment with tunicamycin; (ii) when corrected for this value, the percentage inhibition of GlcN incorporation into various glycoconjugates by tunicamycin in these cells was 82% for glycoproteins, 54% for neutral glycosphingolipids, and 50% for gangliosides; and (iii) the different gangliosides were affected differentially, with the most striking inhibition apparent in GM(3) biosynthesis, which was decreased 78% in the presence of tunicamycin. These data suggest that the effects of tunicamycin on glycosphingolipids as well as on glycoproteins must be considered when interpreting its effects on intact cells and organisms.

Acetylglucosamine↗
PubMed1983

The spectrum of human lung cancer cells in culture: a potential model for studying molecular determinants of tumor progression and metastasis.

We have reviewed the behavior of human lung cancer cells in culture. We have considered the implications of cell biology and biochemistry studies of the cultured cells for the in vivo behavior of lung neoplasms, including the metastatic potential of the different tumor types. Among the human lung cancers, SCC, which grows in culture as suspended aggregates of adherent cells, metastasizes earlier in the life cycle of the tumor and more widely than the major types of non-SCC lung cancers. These latter tumors grow as anchorage-dependent cells in culture. We have pointed out that ultrastructural studies of cultured human lung cancer cells reveal dramatic changes in cell surface membrane morphology coincident with progressive loss of SCC features and intercellular adhesion. We have stressed the potential value of these cultures of human lung cancer cells for studying the molecular determinants of intercellular adhesion and their importance to the process of metastasis. In this context, we have stressed that a different cell surface protein phenotype exists between SCC and non-SCC lung cancer cells in culture and that some of these proteins could be intimately involved in the different growth behavior patterns of these tumor cells. Finally, we have pointed out the need to construct models in which the growth characteristics and biochemical properties of cultured lung cancer cells can be directly compared in vivo with their metastatic potential.

Carcinoma, Small Cell↗
PubMed1983

The relationship between humoral stimulating activity and colony stimulating factor.

Humoral stimulatory activity (HSA) is detected in the sera of neutropenic rodents and humans by its effects on in vitro marrow cell [3H] TdR incorporation. Increased levels of CSA are also found in such sera; however, the relationship between these two activities has not been established. To examine this question, sera were obtained from mice, rats and humans at the peak occurrence of HSA following cyclophosphamide administration and were incubated with rabbit antiserum directed against mouse L-cell CSF. Anti-CSF neutralized the HSA effect on species-specific marrow cell [3H] TdR incorporation relative to the effects of normal sera with an efficiency of anti-CSF produced graded neutralization of the human HSA effect on human marrow cell proliferation. This neutralizing effect of anti-CSF on human HSA was obrogated by prior incubation of anti-CSF with sheep-anti-rabbit-IgG (S-anti-R-IgG) serum. The human serum activities on marrow cell proliferation, as determined by the [3H] TdR incorporation assay, correlated closely with their effects on human granulocyte colony formation in agar culture. These data indicate that human and rodent drug-induced HSA are cross-reactive with CSF as measured by a simple proliferation assay.

Animals↗
PubMed1982

A feasibility study in the development of biological markers for ovarian cancer.

As a primary feasibility study for the selection of biomarkers for more extended clinical evaluation, 17 potential biomarker candidates were measured in the body fluids of patients with ovarian carcinoma. For comparative purposes, patients were staged and separated into three groups: those considered completely free of disease, those with residual but minimal tumor, and those with advanced disease. Individual markers included plasma carcinoembryonic antigen, serum human chorionic gonadotrophin, urinary beta-aminoisobutyric acid, serum UDP-galactosyltransferase, and urinary hydroxyproline. Structurally related groups of biomarkers included six modified nucleosides and three polyamines analyzed in urine, and three serum protein-bound neutral carbohydrates. The respective chromatographic methods developed for these latter biochemical materials enabled all the individual compounds in each group to be quantitated simultaneously in one analytical run. The general frequency and degree of elevation for the total number of biomarkers was directly proportional to increasing tumor burden with specific exceptions, human chorionic gonadotropin and beta-aminoisobutyric acid. Galactosyltransferase was the most frequently elevated in the limited disease categories. Several of the biomarkers were elevated in the majority of patients with advanced disease and appeared potentially superior to carcinoembryonic antigen or human chorionic gonadotrophin.

Aminoisobutyric Acids↗
PubMed1982

A unique cell-surface protein phenotype distinguishes human small-cell from non-small-cell lung cancer.

We have used radioiodination (125I) and two-dimensional polyacrylamide gel electrophoresis to determine that small- (oat) cell lung carcinoma (SCC)--a tumor with neuroendocrine features--possesses a surface protein pattern distinct from the other types of lung cancer cells (squamous, adeno-, and large-cell undifferentiated carcinoma). Twelve distinguishing proteins, 40 to 70 kilodaltons (kDal), characterized four separate lines of SCC; three of these, designated E (60 kDal; pI = 7.3), S (30 kDal; pI = 6.0), and U (57 kDal; pI = 5.6), may be unique SCC gene products and were identified only in [35S]methionine labeling of SCC and not in non-SCC or human fibroblasts. Two lines of adeno-, one of squamous, and one of undifferentiated large-cell lung carcinoma exhibited similar surface protein patterns to one another. Nine distinguishing proteins (40 to 100 kDal) and at least five large proteins (greater than 100 kDal) were unique to these lines. The surface protein phenotypes for SCC and non-SCC were distinct from those for human lymphoblastoid cells and fibroblasts. However, the neuroendocrine features of SCC were further substantiated because 6 of the 12 distinguishing SCC surface proteins, including E and U, were identified on human neuroblastoma cells. The proteins identified should (i) help define differentiation steps for normal and neoplastic bronchial epithelial cells, (ii) prove useful in better classifying lung cancers, and (iii) be instrumental in tracing formation of neuroendocrine cells.

Carcinoma, Small Cell↗
PubMed1982

An alternative approach to the quantitation of glucocorticoid-receptor complexes in the nuclei of lymphoid cells.

We have developed a rapid and convenient method for the quantitative assessment of nuclear translocation of glucocorticoid-receptor complexes in lymphoid cells. In this assay, cells are incubated with [3H]dexamethasone at 4 C or 21 C, treated for 15 min at 4 C with the sulfhydryl blocking reagent sodium tetrathionate or methyl methanethiolsulfonate, and then ruptured with the neutral detergent Triton X-100 to separate the nuclei from the cytoplasmic constituents. When cells are incubated with [3H]dexamethasone at 21 C, an average of 45-60% of the specifically bound cellular radiolabel remains with the nuclei. In contrast, less than 15% of the hormone remains with the nuclei when cells are labeled at 4 C. In rat thymocytes, quantitatively similar results were obtained using this new method and a conventional method of isolating nuclei (homogenization of cells in a hypotonic buffer). For both methods, preincubation of the labeled cells with the sulfhydryl blocking reagents was essential to prevent release of the nuclear hormone (or hormone-receptor complex) by a sulfhydryl-dependent labilizing activity apparently located in the cytoplasm. The new method has been successfully used to quantitate uptake of nuclear [3H]dexamethasone-receptor complexes in rat thymocytes, human lymphocytes, and human leukemia cells of lymphoid and myeloid origin. It should prove successful for separating nuclear from cytoplasmic glucocorticoid-receptor complexes in a wide variety of cells, many of which resist disruption by more conventional techniques.

Adrenalectomy↗