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Biomedical subjects

A Tulp

Publications and source records attributed to A Tulp.

At least 37 records · Page 2Linked to original sources
PubMed1994

Major histocompatibility complex class II molecules induce the formation of endocytic MIIC-like structures.

During biosynthesis, major histochompatibility complex class II molecules are transported to the cell surface through a late endocytic multilaminar structure with lysosomal characteristics. This structure did not resemble any of the previously described endosomal compartments and was termed MIIC. We show here that continuous protein synthesis is required for the maintenance of MIIC in B cells. Transfection of class II molecules in human embryonal kidney cells induces the formation of multilaminar endocytic structures that are morphologically analogous to MIIC in B cells. Two lysosomal proteins (CD63 and lamp-1), which are expressed in MIIC of B cells, are also present in the structures induced by expression of major histocompatibility complex class II molecules. Moreover, endocytosed HRP enters the induced structures defining them as endocytic compartments. Exchanging the transmembrane and cytoplasmic tail of the class II alpha and beta chains for that of HLA-B27 does not result in the induction of multilaminar structures, and the chimeric class II molecules are now located in multivesicular structures. This suggests that expression of class II molecules is sufficient to induce the formation of characteristic MIIC-like multilaminar structures.

Animals↗
PubMed1993

Thermostability analysis of major histocompatibility complex class I molecules by temperature gradient gel electrophoresis.

Empty major histocompatibility complex (MHC) class I molecules present on the surface of RMA-S (26 degrees C) cells were loaded with the iodinated peptides APGNYPAL, FAPGNYPAL (SEV-9) and RGYVYQGL (VSV-8), respectively. The thermostability of these peptide-loaded MHC class I molecules was assessed using temperature gradient native polyacrylamide gel electrophoresis. A linear temperature gradient perpendicular to the direction of electrophoresis yielded a graphical representation of the melting of MHC class I molecules. The class I signal disappeared when the peptide melted out of the groove, and gave rise to a second signal due to released peptide. APGNYPAL-loaded class I molecules melted at 11 degrees C with considerable release even at 0 degrees C. VSV-8-loaded class I molecules melted first at 36 degrees C, whereas SEV-9-loaded molecules melted at about 22 degrees C. A discrimination between the binding of SEV-9 to Kb and Db molecules was seen in the melting patterns. Results are discussed in correlation with known crystallographic structures of class I molecules containing peptides in the binding groove.

Amino Acid Sequence↗
PubMed1993

Application of an improved density gradient electrophoresis apparatus to the separation of proteins, cells and subcellular organelles.

A DGE apparatus, made of Perspex, consisting of a separation column (5 x 2.2 cm) and containing a 0-4% linear Ficoll density gradient, was constructed. Only 2.5 cm of the column were used for high resolution separations. A specially designed removable top cone permitted precise gradient introduction, thin sample layering (0.3-1 mm) and precise fractionation after electrophoresis. A bottom circular palladium anode (nongassing) was separated hydrodynamically but not electrically from the density gradient by a cation-permeable membrane. A top circular platinum cathode caused negatively charged particles to migrate upwards (levitation). Thin sample layering permitted short separation times (30-60 min) at only 3 V/cm (10 mA). As for proteins, glycoforms of a1-antitrypsin were separated as well as isoenzymes of beta-hexoseaminidase. Furthermore, separation of transferrin (Tf) from the putative Tf-receptor complex was effectuated. The device was equally suitable for the separation of Megadalton proteins (mucins). Artificial mixtures of intact erythrocytes (rat, rabbit, human) were separated with high resolution. About 10(7) cells (of 100 microns3 cell volume) could be loaded onto the device. Crude microsomes from the human melanoma cell line Mel JuSo were separated after brief trypsin treatment within 38 min at 10 mA. Ratios of the migration velocities of the constituent organelles were: late endosomes (LE):lysosomes (L):Golgi (G):early endosomes (EE) = 1:0.94:0.77:0.55 and under slightly different conditions LE:L:G:endoplasmatic reticulum (ER):plasma membrane (PM) = 1:0.87:0.64:0.58:0.49.

Animals↗
PubMed1986

Construction and analysis of an EMBL-3 phage library containing partially digested human chromosome 21-specific DNA inserts (15-20 kb).

In the mouse-human hybrid cell line SCC 16-5, chromosome 21 is the only human chromosome present. Fractions highly enriched for this chromosome were obtained by applying the chromosome velocity sedimentation technique to this cell line. DNA prepared from these chromosomal fractions was partially digested with Mbo I, size fractionated on an NaCl gradient, and cloned in the EMBL-3 phage vector. The phage library thus prepared was highly enriched for human chromosome 21-specific recombinant DNA sequences 15-20 kb long. Of the approximately 21,000 phage clones obtained, at least 99% were recombinant. Following phage plaque filter hybridization and Southern blotting, it was found that half of the recombinants were positive for human repetitive DNA. Almost all phages harbored highly or middle repetitive human or mouse DNA sequences owing to the large size of the recombinant inserts. In this library, the human chromosome 21 is represented approximately four times. All human recombinants studied thus far contained DNA inserts originating from chromosome 21 only. The employed cloning strategy is discussed with regard to utility, purity, quality, and completeness of chromosome-specific recombinant DNA libraries.

Animals↗
PubMed1986

Inhibition of N-linked oligosaccharide trimming mannosidases blocks human B cell development.

Deoxymannojirimycin (dMM) or swainsonine (SW), which block conversion of high-mannose to complex-type N-linked glycans, strongly inhibited the production of immunoglobulin (Ig) when added to cultures of human lymphocytes together with the polyclonal B cell activators pokeweed mitogen (PWM) and Staphylococcus aureus (SAC). To obtain the inhibitory effect, inhibitor had to be present during the first 36 h of culture. Addition at later timepoints was less effective and showed that neither inhibitor interfered with rate of production or secretion of Ig as such. Viability and proliferation of the lymphocytes, as defined by cell number and rate of DNA synthesis, were not influenced by the presence of dMM or SW, and no changes in the relative number of helper (T4+) or suppressor (T8+) cells were observed. Thus, for normal differentiation of human B lymphocytes into Ig secreting (plasma) cells in response to PWM and SAC, conversion of high-mannose to complex N-linked glycans is essential.

1-Deoxynojirimycin↗
PubMed1984

A separation chamber to sort cells and cell organelles by weak physical forces. V. A sector-shaped chamber and its application to the separation of peripheral blood cells.

A separation chamber of 90 ml effective volume is described, that incorporates a sector shape (to prevent wall sedimentation) and anti-vortex lamellae (to prevent swirling upon acceleration and deceleration) as well as a flow-diverter for undisturbed layering and fractionation. The chamber is run in an ordinary table centrifuge and possesses a resolving power for equilibrium density centrifugation that seems unsurpassed. Peripheral human blood cells were separated in a Percoll gradient yielding in 1 single run the concurrent isolation of 99% pure monocytes, 99% pure lymphocytes and 99% pure granulocytes. Recoveries varied from 94% to 102%. By modifying the shape of the density gradient, either 99% of all monocytes collected were more than 92.5% pure and at the same time separation of small versus large lymphocytes was obtained, or substantial purification of neutrophils and eosinophils was achieved. The potential for simultaneous separation of monocytes, B, T cells and granulocytes was established. Good separations were obtained with buffy coats from 1 U of blood. The chamber also functioned well in the standard Böyum method for mononuclear cell preparation. In addition, a method for the rapid removal of erythrocytes (within 9 min) at unit gravity is described with nearly quantitative yield of white cells.

Cell Separation↗
PubMed1984

Separation and analysis of human chromosomes by combined velocity sedimentation and flow sorting applying single- and dual-laser flow cytometry.

Human chromosomes were separated on basis of size by velocity sedimentation at 52g in a specially designed sedimentation chamber. The chamber has been constructed in such a way that large numbers of chromosomes can be fractionated on a sucrose gradient while wall sedimentation, streaming, and swirling movements of the gradient during centrifugation are eliminated. Flow deflectors in the chamber allow undisturbed introduction and fractionation of the density gradient. The different chromosomal fractions obtained are highly enriched for the various human chromosomes. Individual chromosomes were subsequently sorted to purity by fluorescence activated flow sorting using a FACS IV flow sorter equipped with a 4-W argon-ion laser. Following this procedure, the sorting rate for specific chromosomes can be speeded up by a factor of 5-10 when the preenriched chromosomal fractions are used as starting material. A high chromosome resolution could be obtained by a few simple modifications of the FACS IV. By applying fluorochromes with different DNA-base specificity the sorting possibilities of individual human chromosomes can be improved. In addition, the chromosomal fractions were analysed by dual-laser flow cytometry after staining the chromosomes with Hoechst 33258 and chromomycin A3. In this way the enrichment of virtually all individual human chromosomes in the different chromosomal fractions can be visualized.

Cell Fractionation↗
PubMed1984

Interactions between lymphoid tumor cells and isolated liver endothelial cells.

Interactions were studied between highly metastatic murine MB6A lymphosarcoma cells and rat liver endothelial cells that had been isolated by collagenase perfusion and purified by unit gravity sedimentation. Experiments were performed on the day of isolation. MB6A cells were observed to adhere to the endothelial cells. Addition of rat serum had a striking effect: The endothelial cells spread over the MB6A cell surface, engulfing the tumor cells. The factor involved was nondialyzable and also was present in rat plasma. Similar interactions were seen with highly metastatic ESb and MDAY-D2 lymphoma cells and with nonmetastatic Eb cells. Low-metastatic GRSL 34 leukemia and TA3/Ha ascites mammary carcinoma cells did not adhere to the endothelial cells. With this in vitro model the molecular mechanisms of adhesion to liver endothelium were studied. As a first step, univalent antibodies against MB6A cells were found to inhibit adhesion, indicating involvement of specific cell surface molecules.

Animals↗
PubMed1984

Transient versus permanent expression of cancer-related glycopeptides on normal versus leukemic myeloid cells coinciding with marrow egress.

Release of mature cells from the bone marrow (BM) into the peripheral blood (PB) compartment is supposed to be triggered by changes in cell surface constituents, most probably in glycoproteins. The supposed importance of glycoproteins in marrow exit prompted us to investigate glycopeptides, i.e., the carbohydrate part of the cell-surface-located glycoproteins of isolated human bone marrow cells of the myeloid series at different stages of maturation. Fractionation of cells was performed by a four-step procedure, comprised of density gradient centrifugation and velocity sedimentation at unit gravity in specially designed separation chambers. With this method, promyelocytes/myeloblasts, granulocytes from bone marrow, and granulocytes from peripheral blood were isolated in high quantity with purities up to 90%, 90%, and 100%, respectively. Surface glycopeptides of the various myeloid cells were investigated by gel filtration analysis after metabolic labeling with radioactive fucose or after external labeling with periodate-borotritide under mild conditions. Within the normal myeloid maturation sequence, mature granulocytes within the bone marrow were found to transiently express altered surface glycopeptides, which disappeared after release into the peripheral blood. These oligosaccharide structures appeared similar to those encountered on leukemic blast cells, known as "cancer-related glycopeptides." In contrast to normal granulocytes from BM, leukemic blast cells retained these aberrant carbohydrate structures on their surface after marrow release. A possible role for cancer-related glycopeptides in the process of marrow cell exit might be hypothesized.

Acute Disease↗
PubMed1983

Cytotoxic effects of dexamethasone restricted to noncycling, early G1-phase cells of L1210 leukemia.

The cytostatic and cytolytic effects of dexamethasone were studied as functions of cell cycle position in mouse L1210 leukemia cells. To this end, the cells were separated according to size by sedimentation at unit gravity in a specially designed sedimentation chamber. The fractions were analyzed by radioautography and flow cytophotometry. The size-distributions obtained by 1g sedimentation coincided with cell-cycle age distribution. With increasing fraction number, samples highly enriched in G1, S, and G2/M cells, respectively were obtained: the smallest cells being in early G1 and the largest in mitosis. In the presence of dexamethasone (10(-6)-10(-5) M), growth slowed down after a few cell cycles and the cells accumulated in early G1 phase. Lytic cell kill by continued exposure to the drug was confined to the fractions containing the small, early G1-phase cells. These fractions were also enriched in noncycling cells that were not labeled by prolonged exposure to 3H-thymidine. After removal of dexamethasone, the cells in S and G2/M phase completed cell cycle traverse but were retarded again in the G1 and early S phase of the next division cycle. The data suggest a memory effect for previous drug exposure. It is concluded that the cytostatic and cytolytic effects of dexamethasone are separate, though not unrelated events. Cytolysis is confined to the noncycling cells that in untreated populations can exit from the dividing compartment during a transitional phase of about 60 minutes subsequent to mitotic division. The cytostatic effects potentiate cytolysis by accumulating the cells in the early G1 phase and thus increasing the probability of their transit to the G0 compartment, sensitive for drug-mediated cytolysis.

Animals↗
PubMed1983

Binding and repair of 2-acetylaminofluorene adducts in distinct liver cell populations.

The study of the binding of the liver carcinogen, N-acetyl-2-aminofluorene, to the DNA of the target organ-as the probable initial step in the process of carcinogenesis-has shown that three modes of interaction occur. N-Acetyl-2-aminofluorene is covalently bound with the nitrogen to the carbon 8 of guanine (I) and with the 3-position to the free NH(2)-group of guanine (II). The third mode of interaction is formed by a covalent bond between the nitrogen of 2-aminofluorene and the carbon 8 of guanine (III). In this study the different modes of interaction were measured separately in stromal and parenchymal cells of the rat liver, after a single intraperitoneal dose. The DNA was isolated from nuclei that had been separated by 1g sedimentation. In parenchymal DNA the types of interaction I and III occur in the same amounts one day after application. In stromal cells the amount of interaction I is relatively small and interaction III predominates (ratio III:I = 5). The amount of interaction III in tetraploid hepatocytes (the largest cell population in the studied rats) per mg DNA is about two times higher than in the stromal cells. While the removal of the total amount of DNA-bound carcinogen takes place at the same rate in the two cell types, a difference in rate and efficiency of repair is observed for the different types of interaction. In tetraploid hepatocytes, interaction I is almost completely removed from the DNA 2 weeks after application, while interaction III diminishes to about 1/3 during the first week but the remaining part disappears very slowly. As shown in earlier studies, interaction II remains in the DNA at a constant level.

2-Acetylaminofluorene↗