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Biomedical subjects

H A Kaplan

Publications and source records attributed to H A Kaplan.

At least 19 recordsLinked to original sources
PubMed2001

Rapid imaging of human melanoma xenografts using an scFv fragment of the human monoclonal antibody H11 labelled with 111In.

H11 is a human IgM monoclonal antibody which recognizes a novel tumour-associated antigen expressed on melanoma, glioma, breast cancer, colon cancer, prostate cancer, lung cancer and B-cell lymphoma. In this study, a recombinant single-chain Fv (scFv) fragment of H11 labelled with 111In was investigated for tumour imaging in athymic mice implanted subcutaneously with A-375 human melanoma xenografts. H11 scFv was derivatized with diethylenetriaminepentaacetic acid (DTPA) for labelling with 111In. The immunoreactivity of DTPA-H11 scFv against A-375 cells in vitro ranged from 23% to 36%. 111In-DTPA-H11 scFv was rapidly eliminated from the blood and most normal tissues (except the kidneys) reaching maximum tumour/blood ratios of 12:1 at 48 h post-injection. Tumours were imaged as early as 40 min after injection. The kidneys accumulated the highest concentration of radioactivity (up to 185% injected dose/g). Tumour uptake was 1-3% injected dose/g. The whole-body radiation absorbed dose predicted for administration of 185 MBq of 111In-DTPA-H11 scFv to humans was 37 mSv. The radiation absorbed dose estimates for the kidneys, spleen and intestines were 405 mSv, 698 mSv and 412 mSv, respectively. The results of this preclinical study and a concurrent phase I trial suggest a promising role for H11 scFv for tumour imaging.

Animals↗
PubMed1997

Immunoreactivity of human MAb BT32/A6 with neuroepithelial tumors.

The present study was undertaken to determine the pattern of immunoreactivity of BT32/A6, a human IgM monoclonal antibody (MAb), with the following histological panels: 1) 30 human and non-human cell lines, 2) 32 normal human tissues, and 3) 28 tumors of central neuroepithelial origin (16 astrocytic; 11 non-astrocytic). Antibody BT32/A6 recognizes a surface and cytoplasmic antigen present on a variety of human tumor cell lines including gliomas, melanomas, neuroblastomas, and a few sarcomas. The antigen is present (at least focally) on 15/16 astrocytic tumor tissue sections (94%), and in some cases, on close to 100% of cells. All malignant cell types, including small anaplastic cells, giant cells, gemistocytic cells, and cells forming pseudopalisades were labeled by MAb BT32/A6. Non-astrocytic neuroepithelial tumors did not stain appreciably with MAb BT32/A6. There was weak immunoreactivity in a small subset of normal human tissues of epithelial and lymphoid origin, with the exception of adrenal cortex, which exhibited weak to moderate staining. All normal tissues of neuroectodermal and mesenchymal origin were unreactive. In conclusion, MAb BT32/A6 appears to be unique in that it recognizes a highly-expressed astrocytic tumor-associated antigen that is present on both low and high grade tumors. This makes it a strong candidate for further studies aimed at establishing its usefulness in the treatment of human astrocytic tumors.

Antibodies, Monoclonal↗
PubMed1995

Human monoclonal antibody BT32/A6 and a cell cycle-independent glioma-associated surface antigen.

The purpose of this study was to ascertain how various growth parameters may influence the labeling of SK-MG-1, a human glioma cell line, by BT32/A6, a human immunoglobulin M monoclonal antibody (MAb). By growing SK-MG-1 cells at different culture split ratios, significant trends in cell growth rate, culture viability, and cell cycle state were produced. Labeling of SK-MG-1 cells by BT32/A6, however, was shown to be unaffected by culture split ratio (p > 0.05) and is therefore independent of cell growth rate, culture viability, and cell cycle state. Using flow cytometry and fluorescence-activated cell sorting, BT32/A6 was shown to label a cell surface antigen on viable, clonogenic cells of SK-MG-1. Approximately 100% of SK-MG-1 cells were shown by flow cytometry to express the BT32/A6 antigen. The recognition of a glioma-associated, cell cycle-independent surface antigen by MAb BT32/A6 makes it a promising candidate for further studies aimed at elucidating its usefulness as an adjunct in the treatment of human malignant gliomas.

Antibodies, Monoclonal↗
PubMed1991

Glycosylation site binding protein and protein disulfide isomerase are identical and essential for cell viability in yeast.

Glycosylation site binding protein (GSBP) has been shown to be identical to protein disulfide isomerase (PDI; EC 5.3.4.1) in a variety of multicellular organisms. We have utilized immunological and biochemical techniques to determine if GSBP and PDI are identical in yeast. Antiserum prepared against yeast GSBP identified in microsomes by its ability to be labeled with a peptide photoaffinity probe was found to recognize PDI purified from yeast. Moreover, this purified yeast PDI was found to be specifically labeled by the photoaffinity probe originally used to identify GSBP in a variety of eukaryotes. On the basis of these observations, we conclude that yeast GSBP and PDI are the same protein. The structure of the yeast PDI gene revealed a product with sequence similarity to higher eukaryotic PDI/GSBP. Disruption of this gene in yeast resulted in a recessive lethal mutation, indicating that PDI/GSBP is required for cell viability.

Affinity Labels↗
PubMed1991

Glycosylation site-binding protein is not required for N-linked glycoprotein synthesis.

In prior studies we identified a 57-kDa protein in the lumen of the endoplasmic reticulum that, in addition to having both protein disulfide isomerase and thyroid hormone-binding protein activities, bound a photoaffinity probe containing the N-glycosylation-site sequence Asn-Xaa-Ser/Thr. It was hypothesized that this multifunctional protein, called glycosylation site-binding protein (GSBP), participated in the process of N-glycosylation of proteins. To test this hypothesis we have employed various conditions to deplete the lumen of GSBP and then assess the level of N-glycosylation catalyzed by oligosaccharyltransferase (OTase). Although most conditions leading to depletion resulted in partial loss of OTase activity, this loss was independent of the extent of GSBP depletion. Indeed, virtually complete loss (greater than 99%) of GSBP with partial retention of OTase activity was frequently observed. Moreover, repletion of the microsomal lumen with GSBP did not restore OTase activity to control levels. Thus, no correlation between GSBP content and OTase activity before or after reconstitution was found. These results suggest that this multifunctional 57-kDa protein is not an essential component of the enzymatic reaction in which oligosaccharide chains are transferred from dolichyl-P-P-GlcNAc2Man9Glc3 to nascent polypeptides or to synthetic tripeptide acceptors.

Animals↗
PubMed1988

Glycosylation site binding protein, a component of oligosaccharyl transferase, is highly similar to three other 57 kd luminal proteins of the ER.

A 57 kd component of oligosaccharyl transferase, termed glycosylation site binding protein, specifically recognizes a photoaffinity probe containing the N-glycosylation site sequence Asn-Lys-Thr. It is present in the lumen of the ER (endoplasmic reticulum) and its release from this compartment results in a loss of N-glycosylation. Antibodies against this protein were used to identify cDNA clones from a lambda gt11 expression library. Analysis of its cDNA sequence reveals high sequence similarity to three other 57 kd luminal endoplasmic reticulum proteins: protein disulfide isomerase, the beta-subunit of prolyl hydroxylase, and thyroid hormone binding protein. This finding suggests that the capacity to recognize multiple polypeptide domains may reside in a single luminal protein that participates in co- and/or posttranslational modifications of newly synthesized proteins.

Amino Acid Sequence↗
PubMed1988

Partial characterization and purification of the glycosylation site recognition component of oligosaccharyltransferase.

Oligosaccharyltransferase, the enzyme catalyzing the co-translational transfer of oligosaccharide from dolichyl-PP-GlcNAc2Man9Glc3 to -Asn-X-Ser/Thr- sequences in nascent polypeptide chains, was studied in hen oviduct microsomes using the active site-directed photoaffinity probe 125I-labeled N alpha-3-(4-hydroxyphenylpropionyl)-Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2. Several lines of evidence established that the tripeptide probe interacted with a 57-kDa protein of the endoplasmic reticulum that was subsequently glycosylated and converted to a 60-kDa form. The 57-kDa protein, isolated by two-dimensional gel electrophoresis, was used as immunogen to prepare polyclonal antisera. The specificity of the antibody was established on the basis of its ability to 1) recognize the 57-kDa protein by immunoblotting and 2) immunoprecipitate the photolabeled protein. The antibody also recognized photolabeled protein from different tissues and organisms. The 57-kDa protein isolated by immunoprecipitation retained its ability to interact with the photoaffinity probe but was inactive in catalyzing glycosylation of peptides. This result suggests that the 57-kDa protein is the component of oligosaccharyltransferase that recognizes the glycosylation site in polypeptides. These results are discussed in terms of possible models for the structure of oligosaccharyltransferase in the endoplasmic reticulum.

Affinity Labels↗
PubMed1988

Estrogen regulation of the central enzymes involved in O- and N-linked glycoprotein assembly in the developing and the adult rabbit endocervix.

During cervical differentiation in 1- to 6-month-old rabbits, a marked increase was observed in the titer of serum estradiol, the number of secretory cells in the endocervix, and the granule content of these cells. Because these secretory granules are rich in carbohydrates it seemed likely that hormones regulate glycoconjugate biosynthesis in the endocervix. To investigate this possibility, the synthesis of O- and N-linked glycoproteins was studied in cell-free preparations from endocervical epithelium. The activity of N-acetylgalactosaminyl (GalNAc) transferase, the first enzyme in the pathway for O-linked oligosaccharide chain biosynthesis, measured in microsomes prepared from the developing cervix, was increased 8-fold. Oligosaccharyltransferase, the enzyme that catalyzes the first step in the attachment of N-linked oligosaccharide chains to proteins, was measured in microsomes prepared under the same conditions as those used for GalNAc transferase. The results of two independent assay methods revealed an estrogen-dependent 10- to 15-fold increase in oligosaccharyltransferase during cervical differentiation. Consistent with these developmental effects, ovariectomy of adult rabbits resulted in reduced (P less than 0.01) titers of serum estradiol and a 2-fold reduction in the specific activity of GalNAc transferase. When animals were treated with exogenous estradiol, GalNAc transferase activity returned to estrous control levels. The antagonistic action of progesterone on GalNAc transferase activity was verified using endocervical membranes from pseudopregnant animals. Similarly, oligosaccharyltransferase activity was reduced 2- to 3-fold when estrous animals were ovariectomized or made pseudopregnant. The treatment of ovariectomized animals with estradiol resulted in the restoration of oligosaccharyltransferase to estrous control values. Collectively, these results provide the first definitive evidence that hormones can regulate the activity of the enzymes involved in the attachment of O- and N-linked oligosaccharide chains to proteins in the endocervix.

Aging↗
PubMed1987

The psychopathology of nostalgia.

Nostalgia is distinguished from depression, of which it was originally considered a variant. It is described as a pleasurable affect involving warm memories of the past. For nostalgia to be normal, it must contain a depressive component that is related to the recognition that the past is irrevocable. In its pathological form, the mood contains only the elated aspects without the acceptance of loss, or what could be described as bittersweet sentiment. The pathological form serves mainly denial and functions like a screen affect. I have attempted to show how particular developments play a role in pathological nostalgia, namely, screen affects, ego ideal formation, and rescue and family romance fantasies. The patients that were described enlisted their nostalgic pursuits as a way of remaining close to the past. These pursuits served both a defensive function, as a way of avoiding the humiliation of oedipal and later defeats, as well as offering them instinctual gratification through fantasy.

Adult↗
PubMed1986

Studies on properties of membrane-associated oligosaccharyltransferase using an active site-directed photoaffinity probe.

Previous attempts in several laboratories, including ours, to purify oligosaccharyl-transferase have met with limited success because of the lability of the membrane-associated enzyme after solubilization with detergents. In an effort to identify the enzyme in face of this lability, we recently developed a photoaffinity reagent to label the active site [J. K. Welply, P. Shenbagamurthi, F. Naider, H. R. Park, and W. J. Lennarz (1985) J. Biol. Chem. 260, 6459-6465]. In this report, the preparations of a more sensitive selective labeling probe, 125I-labeled N alpha-3-(4-hydroxyphenylpropionyl)-Asn-Lys-(N epsilon-p-azidobenzoyl)-Thr-NH2, is described. Using this new probe, we have confirmed, independently of catalytic activity, that hen oviduct oligosaccharyltransferase is tightly associated with the endoplasmic reticulum membrane. The 125I-labeled oligosaccharyltransferase was released from the membrane by detergent and strong alkali treatments but not by sonication, high salt, or hypotonic shock. However, all procedures that released the enzyme from the membrane resulted in a dramatic loss of enzyme activity. Treatment of sealed microsomal membrane vesicles with phospholipase A resulted in nearly complete enzyme inactivation; in contrast, phospholipase C or D had moderate or little effect, respectively. Taken together, these results suggest that the hydrophobic environment of the membrane is required for oligosaccharyltransferase activity. Trypsin treatment of intact vesicles diminished enzyme activity by nearly 70%, but it had no effect on the binding affinity of the enzyme for the 125I-labeled photoaffinity probe. This result suggests that the polypeptide acceptor portion of oligosaccharyltransferase is lumenally disposed, and that a trypsin-sensitive, cytoplasmically oriented domain or another subunit binds the carbohydrate donor, dolichol-PP-oligosaccharide.

Affinity Labels↗
PubMed1986

N-linked glycoprotein biosynthesis in the developing mouse embryo.

We have developed microenzymic assays that have, for the first time, enabled analysis of several enzymes in the pathway for N-linked glycoprotein biosynthesis in pre- and peri-implantation mouse embryos. The in vitro activities of the glycosyl transferases responsible for the formation of N-acetylglucosaminylpyrophosphoryldolichol,N, N'-diacetyl-chitobiosylpyrophosphoryldolichol, mannosylphosphoryldolichol, and glucosylphosphoryldolichol were found to decrease after fertilization before increasing significantly at the blastocyst stage, a stage that was also found to be highly sensitive to the glycosylation inhibitor, tunicamycin. The observed elevation in the activities of these enzymes in blastocysts still occurred when ebbryos were cultured in alpha-amanitin, indicating that de novo mRNA synthesis is unnecessary for the observed increase in their activities. Thus, an elevated capacity for N-glycosylation exists at the blastocyst stage, a time when dramatic increases in cell-cell interactions are known to occur.

Amanitins↗
PubMed1986

Fibronectin and laminin promote in vitro attachment and outgrowth of mouse blastocysts.

The process of mammalian implantation has been investigated using an in vitro model system wherein the trophoblast cells of mouse blastocysts attach to and outgrowth on tissue culture plates containing a complex medium. We now report that two extracellular matrix glycoproteins, fibronectin and laminin, when individually precoated on tissue culture plates promoted in vitro attachment and outgrowth of mouse blastocysts in serum-free medium. The kinetics of attachment and outgrowth processes in the presence of either of these two proteins were identical to that observed in complex, serum-containing medium. In contrast, plates containing a collagen matrix or pretreated with a variety of other serum proteins or various lectins failed to support in vitro attachment and outgrowth of blastocysts. Because all components of the culture medium are defined and both fibronectin and laminin are known components of the basement membrane of the endometrium, this in vitro system offers considerable advantages over the serum supplemented system to study in vitro implantation.

Animals↗
PubMed1986

The effect of hexapeptides on attachment and outgrowth of mouse blastocysts cultured in vitro: evidence for the involvement of the cell recognition tripeptide Arg-Gly-Asp.

Differentiation of the nonadherent trophectoderm cells of the mammalian embryo into attachment-competent trophoblast cells appears to be a prerequisite to invasion of the uterine stroma. To investigate the molecular basis of trophoblast differentiation free of maternal environmental constraints, we used a model system in which attachment and outgrowth of trophoblast cells occurs in vitro. Recently, it was found that either fibronectin or laminin, both of which are extracellular matrix glycoproteins of the uterine stroma, will support trophoblast outgrowth in vitro. In this study we report that the outgrowth of blastocysts on fibronectin-coated dishes is inhibited in a dose-dependent manner by the presence of a hexapeptide containing the sequence Arg-Gly-Asp, which has been shown previously to be recognized by the fibronectin receptor. This peptide had no effect on laminin-mediated trophoblast outgrowth, suggesting that the trophoblasts contain different cell surface receptors for fibronectin and laminin. Trophoblast attachment and limited outgrowth also could be obtained on dishes to which the hexapeptide Gly-Arg-Gly-Asp-Ser-Pro was coupled. Under these conditions, however, outward migration of the trophoblast cells appeared to be reduced. Vitronectin, another adhesion molecule that apparently binds to cells via a cell surface receptor that recognizes Arg-Gly-Asp sequences, also was capable of supporting trophoblast outgrowth. These findings suggest that differentiation of cells of the trophectoderm into trophoblast cells with an invasive phenotype may involve the production of cell surface receptors for fibronectin and possibly for other proteins that contain the Arg-Gly-Asp recognition sequence.

Amino Acid Sequence↗
PubMed1984

Studies of the effect of experimental inflammation on rat liver nucleotide sugar pools.

The effect of experimental inflammation on levels of nucleotide sugars was studied in rat liver. There was an increase of about 2-fold in the levels of UDP-N-acetylhexosamines and GDP-Man at 8 and 4 hr after inflammation, respectively. At 48 hr after inflammation GDP-Man had returned close to control values, but UDP-N-acetylhexosamines were still about 50% above controls. There was a 30% reduction in CMP-NeuAc and UDP-Gal at 8-12 hr after inflammation before increasing to slightly above controls at 16-48 hr after inflammation. Inflammation resulted in an increase in activities of glucosamine-6-phosphate synthase and UDP-GlcNAc-2'-epimerase to about twice control activities at 24 and 8 hr after inflammation, respectively, before declining; CMP-NeuAc synthase activities did not show large changes following inflammation.

Acetylgalactosamine↗