Biomedical subjects

R G Ulrich

Publications and source records attributed to R G Ulrich.

At least 73 records · Page 4Linked to original sources

PubMed1988

Acute hepatocellular effects of erythromycin, gentamicin, and trospectomycin in the perfused rat liver: lack of correlation between lamellar body induction potency and cytotoxicity.

The formation of multilamellar inclusion bodies in cytoplasm is a generalized cellular response to treatment with a variety of chemical agents. The present study was conducted to determine if a correlation exists between acute lamellar body induction potency and cytotoxicity in the perfused rat liver. Livers were perfused for 3 hrs with various concentrations of erythromycin, gentamicin, sulfate, or trospectomycin sulfate, all of which are known to produce lamellar bodies in the rat in vivo. At the end of the experiments, the livers were perfusion fixed for transmission electron microscopy. Based on the bile flow rate, perfusion rate at constant pressure, and cytoplasmic enzyme release, neither gentamicin nor trospectomycin was hepatotoxic at concentrations up to 1.8 mM, whereas erythromycin was toxic at 0.1 mM. Gentamicin caused no ultrastructural changes compared to controls, but trospectomycin caused the dose-dependent formation of lamellar bodies in hepatocytes without other cytoplasmic alterations. Erythromycin caused cellular degeneration accompanied by an increase in the number of secondary lysosomes, but these lacked lamellated inclusions. It is concluded that hepatic lamellar bodies can be induced in acute ex vivo experiments, but that their formation does not appear to be linked with acute cytotoxicity.

Animals↗

PubMed1988

Augmentation of serum bactericidal activity by paldimycin.

At concentrations below the MIC, paldimycin induced changes in Staphylococcus aureus 502A (UC 9116, ATCC 28417) which increased its sensitivity to serum. The enhanced sensitivity to serum was concentration dependent with the maximal sensitivity found when bacteria were grown in approximately 1/10 MIC of paldimycin. Within an 1-hour incubation, S. aureus 502A typically grew 1.5-2-fold in serum. Following exposure to paldimycin, however, approximately 30-50% of the bacteria were killed in serum. The paldimycin treated bacteria were not more susceptible to phagocytosis and killing by polymorphonuclear leukocytes. At the concentrations utilized, the Staphylococci were enlarged and had thickened cell walls. The organisms were still viable and replicating, but irregularities in cell division were observed in transmission electron micrographs.

Acetylcysteine↗

PubMed1987

Immune recognition of human major histocompatibility antigens: localization by a comprehensive synthetic strategy of the continuous antigenic sites in the first domain of HLA-DR2 beta chain.

A comprehensive synthetic approach, previously developed in this laboratory, has been applied to screen the entire first domain (residues 1-95) of the HLA-DR2 beta chain for the full profile of continuous regions that are recognized by human allo-antisera and anti-DR or anti-beta chain antisera raised in other species. Nine consecutive peptides, that were 15 residues each and overlapped by 5 residues, covering the first domain of the DR2 beta chain were synthesized, purified and characterized. The antibody-binding activities of the peptides were determined by radioimmunosorbent titrations. This established the full profile of peptides having specific antibody-binding activity with these various antisera. Three continuous antigenic sites were localized in this domain by all of antisera tested. Peptide 81-95 appeared to be the most immunodominant (i.e. bound the highest amounts of antibodies) in most antisera tested. Although their immunodominance varied with the antisera and boundary shifts were present, the submolecular regions recognized on the first domain of DR2 beta appeared to be similar with antisera against the DR2 (alpha beta I) complex or against the isolated beta chain. Furthermore, recognition was independent of the host species from which the antisera were obtained.

Animals↗

PubMed1987

The continuous antigenic regions in the second domain of the beta chain of human MHC DR2 antigen: antigenic profile of the entire extracellular part of the chain.

Ten consecutive 15-residue peptides (except for peptide 181-198) that spanned the entire second domain of the HLA DR2 beta chain and overlapped by 5 residues were synthesized, purified and characterized. The antibody-binding activities of the peptides with human alloantisera and with antisera raised in other species were determined by radioimmunosorbent titrations. This established the full profile of peptides having specific antibody-binding activity with these various antisera. The continuous antigenic sites in this domain were localized within four regions. The localization of the antigenic regions on the second domain completes the description of the antigenic profile of the entire extracellular part of the beta chain of HLA DR2. Thus, the continuous antigenic sites on the extracellular part of the DR2 beta subunit reside within seven regions with an eighth site being recognized somewhat weakly with some antisera. As was found for the first domain, the immunodominance of a given site varied with the antisera and boundary shifts were present. The submolecular regions recognized on the second domain of the DR2 beta were independent of the host species from which the antisera were obtained.

Amino Acid Sequence↗

PubMed1987

Erythrocyte-mediated microinjection of horseradish peroxidase into BHK cells. Subcellular localization by light and electron microscopy.

In order to establish the distribution with time of proteins microinjected into mammalian cells, horseradish peroxidase (HRP) was microinjected into baby hamster kidney (BHK) cells using chicken erythrocyte ghosts. At time intervals following initiation of fusion between ghosts and target cells, samples were fixed with aldehydes and the peroxidase visualized by reaction with diaminobenzidine and viewing by light and electron microscopy. At 10 min, the reaction product was observed within the cytoplasm of 60% of the microinjected cells, but was excluded from the nucleus and membranous organelles. In the other 40% of microinjected cells, the reaction product was also observed within the nucleus. At 30 min, the reaction product was observed to be evenly distributed throughout the cell, including the nucleus but excluded from organelles. By 6 h, the reaction product was present almost exclusively within the nucleus of 63% of microinjected cells. At all time points, 20-30% of the erythrocytes ghosts appear to have been taken up by cells by phagocytosis rather than fusion, as evidenced by the presence of peroxidase reaction product within intact and fragmented erythrocyte ghosts in the cytoplasm of target cells. Cells incubated with a lanthanum solution following fusion excluded this electron dense tracer, indicating that the cytoplasmic compartment is not opened during exposure to polyethylene glycol.

Animals↗

PubMed1987

Folding and activation of recombinant human prorenin.

In vitro folding of mature renin, prorenin, and fused prorenin, all produced in denatured form in inclusion bodies in recombinant Escherichia coli, has been studied in order to evaluate the importance of prosequence in the folding of human renin. These studies have been compared with the in vivo folding and subsequent in vitro activation of recombinant human prorenin secreted by a nonbacterial expression system, namely Chinese hamster ovary (CHO) cells grown in serum-free medium. It is concluded that prosequence is essential in the folding of human renin and, therefore, the DNA coding for this sequence cannot be removed without affecting the recovery of active human renin from recombinant bacterial and nonbacterial systems.

Cell Line↗

PubMed1986

Interaction of monoclonal antibodies directed against bromodeoxyuridine with pyrimidine bases, nucleosides, and DNA.

Although antibodies directed against bromodeoxyuridine (BrdU) are being used in both clinical and basic research laboratories as tools to study and monitor DNA synthesis, little is known about the epitopes with which they react. Four monoclonal antibodies directed against BrdU were produced and were characterized to learn more about the epitopes on BrdU which are important for antibody recognition, to identify compounds other than BrdU which react with the antibodies and which might interfere with immunologic assays for BrdU, and to characterize the reaction of these antibodies with BrdU-containing DNA. By radioimmunoassays, the antibodies generally reacted well with 5-iododeoxyuridine, 5-fluorodeoxyuridine, and 5-nitrouracil. However, none of the antibodies reacted well with uridine--indicating that a substituent on uridine C5 was essential for antibody reactivity--or with 5-bromo- or iodo-cytosine, indicating that the region around pyrimidine C4 is important for antibody recognition. Although the antibodies reacted with 5-halogen-substituted uracil bases, the antibodies reacted much better with the corresponding halogenated nucleosides, indicating that the sugar moiety was important for recognition. The presence of a triphosphate group on C'5 of BrdU (i.e., BrdUTP) did not detectably alter antibody recognition. Three of the antibodies reacted only with purified DNA containing BrdU, whereas one antibody, which exhibited a weak interaction with thymidine, also reacted with BrdU-free DNA. S1 nuclease treatment of purified DNA suggested that all four monoclonal antibodies reacted exclusively with single-stranded regions of BrdU-containing DNA. Comparison of detecting DNA synthesis by [3H]TdR incorporation followed by autoradiography with that by BrdU incorporation followed by indirect immunofluorescence indicated that the latter technique was both an accurate and a sensitive measure of DNA synthesis.

Animals↗

PubMed1986

Characteristics of aggregated immunoglobulin G as an immunologic phagocytic stimulus for granule enzyme release from human neutrophils.

Aggregated immunoglobulin G (AggIgG) induced a time- and concentration-dependent phagocytic release of granule-associated lysozyme and myeloperoxidase (MPO) from human neutrophils. Degranulation was significantly enhanced in the presence of calcium or magnesium, and maximum granule exocytosis was observed when both divalent cations were present. AggIgG-stimulated enzyme release was inhibited with the intracellular calcium antagonist, TMB-8[8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy)benzoate] in the absence of extracellular calcium. DIDS (4,4'-diisothiocyano-2,2'-disulfonic acid stilbene), a permeant anion channel blocker, also suppressed AggIgG-induced degranulation. Cycloheximide, an inhibitor of protein synthesis, enhanced granule exocytosis from AggIgG-treated neutrophils. Two inhibitors of transmethylation reactions, 3-deazaadenosine (3-DZA) and homocysteine thiolactone (HCTL) in combination, suppressed AggIgG-elicited granule enzyme release. These data indicate that AggIgG is a useful probe for investigating the requirements for phagocytic enzyme release from human neutrophils.

Calcium↗

PubMed1985

Inhibition of DNA replication and DNA polymerase alpha activity by monoclonal anti-(DNA polymerase alpha) immunoglobulin G and F(ab) fragments.

The effect of monoclonal anti-(DNA polymerase alpha) immunoglobulin G (IgG) and F(ab) fragments on DNA replication in lysolecithin-permeabilized human cells and on DNA polymerase alpha activity was determined. DNA polymerase alpha activity in vitro was inhibited equally by the same concentrations of monoclonal IgGs and F(ab) fragments. However, the IgGs and F(ab) fragments were not equally potent in inhibiting DNA replication in permeable cells. In general, the F(ab) fragments were approximately equal to 10-fold more potent than IgGs in inhibiting DNA replication, suggesting the F(ab) fragments cross the nuclear membrane more readily than IgGs. Immunocytochemical studies demonstrated that at least a fraction of anti-(DNA polymerase alpha) IgGs entered the nucleus of permeable cells. For most antibodies tested, the IgG or F(ab) concentration needed to inhibit replication was several orders of magnitude higher than that needed to neutralize polymerase alpha activity extracted from the same number of cells. Anti-(DNA polymerase alpha) F(ab) fragments were shown to inhibit the discontinuous synthesis of Okazaki DNA, as well as the maturation of Okazaki DNA to larger DNA, thereby implicating DNA polymerase alpha in both of these processes.

Animals↗

PubMed1985

Monoclonal antibodies against human DNA polymerase-alpha inhibit DNA replication in permeabilized human cells.

Monoclonal neutralizing antibodies against DNA polymerase-alpha substantially inhibit nuclear DNA replication in lysolecithin-permeabilized cultured human fibroblasts. The degree of inhibition of DNA synthesis is proportional to antibody concentration, and the effect is specific in that RNA synthesis measured under the same experimental conditions is unperturbed. Autoradiographic data demonstrate that the magnitude of the inhibition measured in the mass culture reflects the uniform response of all the constituent cells in the target population. These observations confirm the participation of DNA polymerase-alpha in replicative DNA synthesis and identify a versatile, novel approach to the dissection of mammalian processes of DNA replication and repair.

Antibodies, Monoclonal↗

PubMed1985

Cyclic AMP and theophylline enhance DNA synthesis in L cells stimulated with anti-actin IgG and [(IgG)2 protein A]2 complex by recruiting cells into S-phase.

Surface binding of anti-actin IgG alone or in a Mr = 716 000 [(IgG)2 Protein A]2 complex results in a stimulation of DNA synthesis and cell growth in L cells. Cyclic-AMP (0.01-1.0 mM) added to such cell cultures augmented DNA synthesis as measured by incorporation of [3H]thymidine into DNA. Theophylline (0.1-1.0 mM), a phosphodiesterase inhibitor which prevents enzymatic breakdown of cAMP, had similar effects, but cGMP (0.01-1.0 microM) reversed the effects of cAMP and theophylline upon DNA synthesis. Analysis of the cell cycle by flow cytometry revealed that antibody produced a shift (7%) of cells from the G1-phase to the S-phase (DNA-synthetic) of the cell cycle at 72 hr of incubation. Addition of cAMP (0.5 mM) to cell cultures, however, produced significant shifts of antibody stimulated cells from G1-phase to S-phase at all time points measured, i.e., 24 (12%), 48 (22%), 72 hr (23%). Thus, antibody recruited cells into S-phase of the cell cycle and cAMP greatly augmented the effect. These observations suggest that the mechanism of activation of L cell growth by antibody to surface antigens involves a recruitment of cells into the DNA-synthetic phase and that the effect may be mediated by cAMP.

Actins↗

PubMed1985

Monoclonal anti-actin antibody modulates expression of surface antigen on L cells.

Monoclonal anti-actin antibody which is known to stimulate DNA synthesis and cell growth in a murine transformed cell line, L cell, was examined for its ability to modulate the expression of surface antigen on the cell membrane. There was a time dependent increase in the number of cell surface antigen molecules on L cells stimulated by monoclonal anti-actin antibody as measured by indirect immunofluorescence and flow cytometry. For L cells incubated for 12, 24, 48, 72, and 96 hours with monoclonal anti-actin antibody versus a myeloma control supernatant, the percentage of cells exhibiting high intensity immunofluorescence was 29 vs. 13, 13 vs. 10, 34 vs. 1, 52 vs. 11, and 35 vs. 18, respectively. We conclude that monoclonal anti-actin antibody modulates the time dependent surface expression of actin on L cells. It is likely that this modulated expression of surface actin-like molecules plays an important role in the transmission of stimulatory signals to the cells which result in enhanced cellular metabolism and proliferation.

Actins↗

PubMed1984

Enhanced N-acylation of palmitic acid in sphingomyelin of antibody-stimulated L cells.

Metabolism of sphingomyelin was stimulated in a fibroblast-like transformed cell line, L-929, when the cells were incubated with antibodies of a specific rabbit antiserum. The cells responded with an increased incorporation of [3H] palmitic acid into sphingomyelin. The stimulated uptake of palmitic acid into sphingomyelin can not be explained by simple mass increases in cellular phospholipids but probably represents a selective N-acyl group turnover. Palmitic acid composed only 7.5 percent of the acyl substituents, as assessed by gas-liquid chromatographic analysis, but [3H] palmitic acid was incorporated at a two-fold higher level into the acyl position than into the long chain base precursor (sphingosine). Since it is known that palmitic acid is the predominant fatty acid forming sphingosine, this represents a considerable selection for N-acyl group turnover. Another saturated fatty acid, stearic acid, which was over twice as abundant constituently was incorporated at a much lower rate when the cells were stimulated. Thus palmitic acid was observed to be selectively turning over in a manner suggestive of acylation-reacylation cycles observed with other classes of phospholipids.

Acylation↗

PubMed1984

Incorporation of fatty acids into phospholipids in L cells stimulated by antibody.

Binding antibodies to surface membranes stimulated incorporation of fatty acids (FA) into phospholipids of L cells. Antibodies stimulated at least a 3.4-fold greater incorporation of arachidonic acid into phosphatidylinositol than into any other class of phospholipid when compared on a molar basis (p less than 0.003). This enhanced incorporation was selective, depending on the character of the FA, because antibodies stimulated the incorporation of arachidonic acid at least 2.4-fold more than oleic acid, palmitic acid or stearic acid (p less than 0.001). Surprisingly, an antibody-stimulated incorporation of palmitic acid into sphingomyelin (SM) was at least 2.2-fold greater than that into any other class of phospholipid (p less than 0.001) and the antibody-stimulated incorporation of palmitic acid into SM was at least 60-fold greater than that of arachidonic acid, stearic or oleic acid (p less than 0.001). Nontoxic doses of ethylenediamine tetraacetic acid (EDTA), dexamethasone, 4-bromophenacylbromide and indomethacin inhibited the antibody-stimulated incorporation of arachidonic acid into cellular phospholipids, principally phosphatidylinositol (PI), and similarly inhibited the antibody stimulation of DNA synthesis. We conclude that when antibody binds to surface antigens on L cells, a rapid and selective incorporation of fatty acids into certain cellular phospholipids occurs, possibly mediated by calcium-dependent phospholipases. Degradation products of arachidonic acid, i.e., prostaglandins, may be important in these antibody stimulation events, as well. These early changes in phospholipid metabolism may serve as an important signal or mechanism for the subsequent stimulation of DNA synthesis in L cells.

Animals↗

PubMed1984

Antibody to immunoselected L-cell antigens mimics stimulating activity of antibody to whole L cells.

A heterogeneous IgG antibody raised in rabbits in response to injections of whole L cells was used to identify and select relevant antigens in a nonionic detergent extract of L cells prelabeled with [35S]methionine by means of immunoprecipitation and immunoaffinity chromatography. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the immunoprecipitate and immunoeluate contained far fewer protein bands than the whole cell extract but selectively retained a 42,000-MW protein species. In response to injections of the immunoprecipitate, rabbits produced a new antiserum which reacted predominantly with the 42,000-MW protein when reacted with L-cell proteins separated by sodium dodecyl sulfate-gel electrophoresis and transferred to nitrocellulose paper by the Western blot technique. The new antiserum (raised to the immunoprecipitate) and the original antiserum (raised to whole cells) were equipotent in stimulating calcium transport, phospholipid metabolism, and DNA synthesis in L cells. Binding of the IgG fractions of the two antisera displayed identical high affinity binding to L-cell surface antigens, with the same average association constant of 1.5 X 10(6) M-1. These studies have shown that an antiserum raised to whole L cells has a much narrower reactive spectrum with L-cell membrane antigens than might be imagined and has identified a 42,000-MW membrane protein as an important immunogen which itself elicits a potent immune response resulting in an antibody capable of mimicking the cell stimulatory properties of the original antiserum.

Animals↗

PubMed1984

Subcellular location of stimulus-affected endogenous phosphoproteins in the rat parotid gland.

Rat parotid minces were labeled with [32P]Pi, stimulated with isoproterenol, homogenized in sucrose, and fractionated on continuous sucrose density gradients. We analyzed the resulting fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiograms were made from the gels. Comparison of fractions from control and isoproterenol-stimulated minces revealed seven phosphoproteins that were affected by isoproterenol. The subcellular location of these proteins was determined by comparing their distribution in the sucrose gradients with that of a number of enzymes that are characteristic of specific organelles. Isoproterenol decreased the phosphorylation of two cytoplasmic proteins (Mr 16,000 and 18,000) and increased the phosphorylation of a third (Mr 14,000). The phosphorylation of two endoplasmic reticulum proteins was increased by isoproterenol (Mr 20,500 and 22,500), as was an Mr 31,000 protein which was probably the S6 ribosomal protein. The phosphorylation of a secretory granule protein (Mr 24,000) was decreased by isoproterenol. We then developed a purification scheme for parotid secretory granules. By using this method, we demonstrated that the phosphorylation of the Mr 24,000 was also decreased by carbamylcholine. Granules purified by this method also contained a small number of other phosphoproteins whose phosphorylation was increased only by isoproterenol. Secretory granule-associated stimulus-affected phosphoproteins were found in the particulate fraction when the granules were hypotonically lysed, and were not extracted from the particulate fraction by washing with 0.6 M KCl.

5'-Nucleotidase↗

PubMed1983

Cell surface and cytoskeletal interactions in a temperature-sensitive strain of SV40-transformed mouse fibroblasts.

In order to assess the role of cytoskeletal structure in modulating cell surface topography during cell transformation, cytoskeletal organization of 3T3 mouse cells transformed with a tsA mutant of simian virus 40 (SV40) was studied in detail by correlative light and electron microscopy. Detergent-extracted, critical-point dried whole cells observed in the electron microscope were seen to contain well-organized microfilament bundles (stress fibers) traversing the longitudinal axis of cells grown at the restrictive temperature (39 degrees C). When grown at the permissive temperature (32 degrees C), cells prepared in this manner were not observed to contain such structures. However, when semithin sections (0.5 micron) were viewed by transmission electron microscopy at 120 kV, short microfilament bundles were seen in 32 degrees C-grown cells. There was an alteration in the morphology of these structures at sites of attachment to the substratum (focal contacts), and they were shorter in length than microfilament bundles of 39 degrees C-grown cells. A difference was also observed between the two phenotypes in the layer of microfilaments associated with the dorsal cell surface. Since it is this layer that directly determines cell surface architecture, it is proposed that changes in microfilament bundle-generated surface tension are responsible for alterations of this layer, leading to an altered cell surface morphology. Tension may be modified by disturbances in focal contacts (or adjacent regions) or altered actin-associated protein(s).

Animals↗

PubMed1983

Analysis of erythrocyte-mediated microinjection by backscattered electron imaging.

The process of chicken erythrocyte-mediated microinjection of cultured BHK cells was studied by correlative secondary and backscattered electron imaging. The intense staining of the chicken erythrocyte nucleus by uranyl acetate was found to produce a backscattered electron signal sufficient to follow its position during cell fusion and microinjection. Initially, the erythrocyte ghosts were found to bind to the target cell surfaces. The microinjection process was complete within 2 to 4 hr, as evidenced by the presence of the erythrocyte nucleus within the cytoplasm of the target cell. By 24 hr, the internalized erythrocyte nuclei were difficult to distinguish by backscattered electron imaging. Some erythrocyte ghosts entered the cells intact by some method other than cell fusion, presumably phagocytosis.

Animals↗