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Biomedical subjects

R Peters

Publications and source records attributed to R Peters.

At least 19 recordsLinked to original sources
PubMed1992

Regulation of human calcitonin gene transcription by cyclic AMP.

Transcription of the human calcitonin (CT) gene is markedly increased by cAMP in the TT line of medullary thyroid carcinoma. This response is conferred by 5' flanking DNA sequences located between -132 and -252 relative to the transcription initiation site. Within this region are an upstream cyclic AMP response element (CRE), a downstream CRE flanked by two octamer motifs, and two adjacent C-rich AP2-like sequences. In transfection experiments in TT cells, the downstream CRE, combined with CT promoter sequences, generated 70% of the maximal cAMP response. The upstream CRE and the C-rich elements conferred 10 and 30% of this response, respectively. In gel mobility shift assays, specific TT cell proteins bound to each of these sequences. Therefore, the cAMP response of the CT gene is complex, requiring multiple elements acting in concert.

Base Sequence↗
PubMed1992

Generation of anti-idiotypic monoclonal antibodies recognizing vasopressin receptors in cultured cells and kidney sections.

To produce anti-idiotypic antibodies against receptors for the neurohypophyseal hormone vasopressin, an anti-vasopressin monoclonal antibody with a ligand specificity similar to that of vasopressin receptors was employed for immunization. Three anti-idiotypic monoclonal antibodies were obtained which induced, like vasopressin, plasminogen activator production in the renal epithelial cell line LLC-PK1 (expressing V2-receptors). Induction of plasminogen activator synthesis by the anti-idiotypic antibodies could be inhibited by coincubation with a vasopressin antagonist. In a fashion similar to that of vasopressin itself, the anti-idiotypic antibodies induced receptor down-regulation. The anti-idiotypic antibodies were employed to visualize vasopressin receptors on LLC-PK1 and A7r5 (V1-receptor-expressing) smooth muscle cells by immunofluorescence. Antibody-mediated fluorescence was not observed in receptor-deficient mutant cell lines or vasopressin-receptor-down-regulated cells. Furthermore, these antibodies were used for immunohistochemical localization of vasopressin receptors in rat and bovine kidney preparations. In accordance with earlier physiological and biochemical observations, vasopressin receptors were detected predominantly in collecting ducts in cortex and medulla. On the cellular level, a differential staining pattern was observed.

Animals↗
PubMed1992

Receptor-mediated endocytosis and nuclear transport of a transfecting DNA construct.

A soluble construct consisting of a plasmid carrying the gene of the SV40 large T-antigen and an insulin-poly-L-lysine conjugate is able to selectively transfect PLC/PRF/5 human hepatoma cells which possess insulin receptors. Transfection can be efficiently competed by excess free insulin. To examine intracellular transport of the construct, it was fluorescently labeled and its accumulation on and in cells visualized by video-enhanced microscopy and quantitative confocal laser scanning microscopy. After 2 h at 37 degrees C, the labeled construct was found predominantly in intracellular acidic compartments, with a substantial portion of fluorescence localized both near and in the cell nucleus. Binding, endocytosis, and nuclear localization of the labeled conjugate could all be competed by excess free insulin, thus indicating that entry of the conjugate into cells was specifically mediated by the insulin receptor.

Antigens, Polyomavirus Transforming↗
PubMed1992

A phase I study of WR-2721 in combination with total body irradiation (TBI) in patients with refractory lymphoid malignancies.

This Phase I study was designed to establish the maximum tolerated dose (MTD) of WR-2721 when given twice weekly with total body irradiation (TBI) in the treatment of patients with advanced refractory lymphoid malignancies and to define the toxicities of this combination and schedule. Patients eligible for this study had advanced recurrent indolent non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia (CLL). Patients had symptomatic or progressive disease, a performance status of 0, 1, or 2, and adequate bone marrow, hepatic, and renal function. Only patients failing one or two regimens of prior chemotherapy were eligible. Patients who had received prior extended field irradiation were ineligible. Patients received TBI twice weekly (Tuesday and Friday) to a total of 10 doses at 15 cGy/fx. WR-2721 was given intravenously over 15 min beginning 30 min before irradiation. The escalation of WR-2721 was Level 1: 740 mg/m2 and Level 2: 910 mg/m2. The MTD of WR-2721 was that dose which produced predictable and reversible toxicity and would not interfere with patient well-being. Seven patients were entered onto the study, three at 740 mg/m2 and four at 910 mg/m2. Five patients had CLL and two patients small lymphocytic NHL. No patient had hypotension or nausea requiring reduction in dose level or even interruption of infusion of WR-2721. At 740 mg/m2 no grade 3 or 4 toxicities related to WR-2721 were observed, but two patients could not complete treatment because of TBI-induced prolonged thrombocytopenia following treatments 5 and 8. One patient completed all 10 treatments. At 910 mg/m2 of WR-2721, two patients requested removal from study because of malaise, one after 5 cycles and one after 7 cycles. One patient completed all 10 treatments. One patient was treated with a modified schedule of 7 treatments of 20 cGy/fx and tolerated and completed all treatments but developed significant thrombocytopenia following completion of treatment. No patients had disease progression during treatment. The median survival was 11 months. This study indicates that WR-2721 given at 910 mg/m2 twice weekly with TBI is well tolerated for at least 5 treatments and that 910 mg/m2 of WR-2721 is the MTD with this regimen. In view of the importance of total radiation dose in achieving a response with TBI, a dose escalation study of TBI with 910 mg/m2 of WR-2721 should be performed in patients with indolent non-Hodgkin's lymphoma.

Amifostine↗
PubMed1992

The Fifth Edition of the Addiction Severity Index.

The Addiction Severity Index (ASI) is 12 years old and has been revised to include a new section on family history of alcohol, drug, and psychiatric problems. New items were added in existing sections to assess route of drug administration; additional illegal activities; emotional, physical, and sexual abuse; quality of the recovery environment; and history of close personal relationships. No changes were made in the composite scoring to maintain comparability with previous editions. This article discusses the clinical and research uses of the ASI over the past 12 years, emphasizing some special circumstances that affect its administration. The article then describes the rationale for and description of the changes made in the ASI. The final section provides "normative data" on the composite scores and severity ratings for samples of opiate, alcohol, and cocaine abusers as well as drug abusing inmates, pregnant women, homeless men, and psychiatrically ill substance abusers.

Adolescent↗
PubMed1992

Management of common bile duct stones with a biliary endoprosthesis. Report on 40 cases.

Endoscopic placement of a biliary endoprosthesis has been proposed for the management of choledocholithiasis when stone extraction is difficult or considered hazardous. Over a two year period this approach was used in 40 such patients. There were 24 women and 16 men with a median age of 76 years. In seven patients with severe cholangitis no attempt was made to extract the stones. Twenty three (57.5%) patients underwent a sphincterotomy and four (10%) needle knife papillotomy. The endoprosthesis insertion was considered a temporary measure in 13 (32.5%) patients and definitive treatment in 27 (67.5%). Bile duct drainage was established in all patients. Early complications occurred in six patients (15%), but were without sequelae. Late complications developed in eight (20%) of the patients and included biliary colic (four), cholangitis (three), and cholecystitis (one). Two patients (one cholangitis and one cholecystitis) died as a consequence of the complication. Only patients without a sphincterotomy developed cholangitis. A total of eight patients (20%) underwent surgery (one as an emergency) and nine a repeat endoscopic retrograde cholangio pancreatography (two as an emergency) to clear the duct. The remaining 23 patients are asymptomatic at a median of 13 months (range five to 24 months). Biliary endoprosthesis insertion for choledocholithiasis is an important alternative means of establishing drainage in selected cases, and is probably the optimum method of management for the elderly and or debilitated patients with previous cholecystectomy. Caution must be exercised, however, in patients with an in situ gall bladder.

Adult↗
PubMed1991

Nuclear localization of budgerigar fledgling disease virus capsid protein VP2 is conferred by residues 308-317.

The capsid protein VP2 of budgerigar fledgling disease virus (BFDV) contains two sequences (residues 309-315 and 334-340) which are homologous to the prototypic nuclear localization sequence (NLS) of the simian virus 40 T-antigen. Using recombinant potential NLS-beta-galactosidase fusion proteins we identified amino acid residues 308-317 (VPKRKRKLPT) to be the NLS of BFDV capsid proteins VP2 and VP3. Microfluorometry studies show that the BFDV-VP2 signal is considerably more efficient in nuclear transport kinetics, than the NLS of SV40-VP2, corresponding to amino acid residues 317-326 (PNKKKRKLSR).

Amino Acid Sequence↗
PubMed1991

Cytochrome b5 and a recombinant protein containing the cytochrome b5 hydrophobic domain spontaneously associate with the plasma membranes of cells.

Both cytochrome b5, isolated from rabbit liver microsomes, and LacZ:HP, a recombinant protein consisting of enzymatically active Escherichia coli beta-galactosidase coupled to the C-terminal membrane-anchoring hydrophobic domain of cytochrome b5, were shown to spontaneously associate with the plasma membranes of erythrocytes and 3T3 cells. Association was promoted by low pH values, but proceeded satisfactorily over several hours at physiological pH and temperature. About 150,000 cytochrome b5 molecules or 100,000 LacZ:HP molecules could be associated per erythrocyte. These proteins were not removed from the membrane by extensive washing, even at high ionic strength. After incubation with fluorescently labeled cytochrome b5 or LacZ:HP, cells displayed fluorescent membranes. The lateral mobility of fluorescently labeled cytochrome b5 and LacZ:HP was measured by photo-bleaching techniques. In the plasma membrane of erythrocytes and 3T3 cells, the apparent lateral diffusion coefficient D ranged from 1.0.10(-9) to 8.10(-9) cm2 s-1 with a mobile fraction M between 0.4 and 0.6. The lateral mobility of these proteins closely resembled that reported for lipid-anchored proteins and was much higher than that reported for Band 3, an erythrocyte membrane-spanning protein with a large cytoplasmic domain. These results suggest that the hydrophobic domain of cytochrome b5 could be employed as a universal, laterally mobile membrane anchor to associate a variety of diagnostically and therapeutically useful recombinant proteins with cells.

Animals↗
PubMed1991

Complement pore genesis observed in erythrocyte membranes by fluorescence microscopic single-channel recording.

The formation and opening of single complement pores could be directly observed in erythrocyte ghosts by confocal laser-scanning microscopy employing the recently introduced method of fluorescence microscopic single-channel recording. Resealed sheep erythrocyte ghosts were incubated with human complement. By limiting the concentration of C8, the eighth component of complement, the fraction of cells rendered permeable for the small polar fluorescent probe Lucifer Yellow was varied between 0.50 and 0.90. Under each condition the flux rate, k, of Lucifer Yellow was determined for a substantial number of ghosts. By analysing the sample population distribution of k the flux rate k1 of ghosts with a single pore was found to be (4.8 +/- 1.1) x 10(-3) s-1 consistent with a pore radius of about 3.5 nm (35 A). The genesis of single complement pores was studied by continuous influx measurements while triggering pore formation by a temperature shift. Pore genesis was found to be a very slow process, proceeding on a time scale of several minutes. During pore genesis the influx curves had a sigmoid shape, which excluded the possibility that the pore was preformed on the membrane surface and subsequently inserted. However, the influx curves could be well simulated by a model which assumed that pores grow stepwise by sequential incorporation of C9 monomers. The model predicts conditions under which the incorporation of single monomers can be directly revealed.

Animals↗
PubMed1991

A microassay for the pore-forming activity of complement, perforin, and other cytolytic proteins based on confocal laser scanning microscopy.

A fluorescence microscopic assay for the activity of complement, perforin, and other cytolytic proteins which form transmembrane pores in cellular membranes is described. The assay was worked out and tested with red blood cell membranes (ghosts) and was then applied to intact hemoglobin-free cells. Resealed human erythrocyte ghosts were incubated with complement or perforin. A small polar fluorescent probe (fluorescein-labeled 1-kDa dextran, FD1) which permeates through complement and perforin pores but not through normal cell membranes was added to the samples. The capability of the confocal laser scanning microscope (CLSM) to generate thin optical sections was exploited to visualize and quantitate fluorescence inside single ghosts and thus determine the fraction of ghosts which had become permeable for FD1. The activity of complement or perforin was quantitated by plotting the fraction of permeable cells versus the concentration of the pore-forming protein. The results were in good agreement with those of a conventional hemolytic assay. The CLSM-based assay was then applied to intact hemoglobin-free cells for which only few alternative assays are available. Compared to conventional hemolytic assays for the activity of pore-forming proteins the assay described here can be applied to a large variety of natural and artificial membrane systems. The assay can be performed under nonlysing conditions. Furthermore, the assay is simple, relatively fast, and requires only extremely small amounts of cells and pore-forming proteins.

Animals↗
PubMed1991

Observations after human immunodeficiency virus immunization and challenge of human immunodeficiency virus seropositive and seronegative chimpanzees.

Two human immunodeficiency virus (HIV)-seropositive chimpanzees (A-3 and A-86c) infected 4 yr earlier with HIV, along with one uninfected animal (A-36), were inoculated intramuscularly three times in a year with a gamma-irradiated gp120-depleted HIV immunogen in incomplete Freund's adjuvant. Both previously infected animals promptly developed an anamnestic humoral antibody response after the first dose, and the uninfected animal developed a primary humoral response to the first dose and then an anamnestic response to the second dose. Although HIV had been recovered repeatedly from the seropositive animals, they became persistently virus-culture negative at the time of or just before the first inoculation of the immunogen. Intravenous challenge with 40 chimpanzee-infectious-doses of a heterologous HIV strain (HIVIIIB) was done 4 mo after the third inoculation in the three treated chimpanzees and in an untreated control animal (A-189a). The immunized naive animal (A-36) and the unimmunized control (A-189a) became infected, and virus has been isolated from their peripheral blood mononuclear cells for greater than 2 yr after challenge. However, the two previously infected chimpanzees (A-3 and A-86c) resisted challenge and have remained virus negative by peripheral blood mononuclear cell cocultivation for greater than 2 yr of observation after challenge; moreover, no evidence of reinfection was detectable by PCR. Despite the in vivo resistance, however, peripheral blood mononuclear cells from the resistant animals (A-3, A-86c) remained susceptible to infection by HIV in vitro. These findings reveal that a state of immunity can develop and/or be induced to control and/or prevent HIV infection in the chimpanzees. In the absence of any detectable level of neutralizing antibody in A-3 and a low level in A-86c, the patterns of the responses to challenge seen in the four animals suggest that the cell-mediated immune mechanism must have played a significant role in the resistant chimpanzees both in control of their HIV infection and in their resistance to challenge.

Animals↗
PubMed1991

Functional size of complement and perforin pores compared by confocal laser scanning microscopy and fluorescence microphotolysis.

Confocal laser scanning microscopy and fluorescence microphotolysis (also referred to as fluorescence photobleaching recovery) were employed to study the transport of hydrophilic fluorescent tracers through complement and perforin pores. By optimizing the confocal effect it was possible to determine the exclusion limit of the pores in situ, i.e. without separation of cells and tracer solution. Single-cell flux measurements by fluorescence microphotolysis yielded information on the sample population distribution of flux rates. By these means a direct comparison of complement and perforin pores was made in sheep erythrocyte membranes. In accordance with previous studies employing a variety of different techniques complement pores were found to have a functional radius of approx. 50 A when generated at high complement concentrations. The flux rate distribution indicated that pore size heterogeneity was rather small under these conditions. Perforin pores, generated in sheep erythrocyte membranes at high perforin concentrations, were found to have a functional size very similar to complement pores. Furthermore, the functional size of the perforin pore seemed to be relatively independent of the dynamic properties of the target membrane since in two cell membranes which are very different in this regard, the human erythrocyte membrane and the plasma membrane of erythroleukemic cells, the functional radius of the perforin pore was also close to 50 A. A perforin-specific antibody reduced the functional radius of perforin pores to 45 A.

Animals↗
PubMed1991

The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen.

We have previously demonstrated [Rihs, H.-P. and Peters, R. (1989) EMBO J., 8, 1479-1484] that the nuclear transport of recombinant proteins in which short fragments of the SV40 T-antigen are fused to the amino terminus of Escherichia coli beta-galactosidase is dependent on both the nuclear localization sequence (NLS, T-antigen residues 126-132) and a phosphorylation-site-containing sequence (T-antigen residues 111-125). While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation-site-containing sequence. The present study furthers this observation and examines the role of the various phosphorylation sites. Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry. By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK-II) site S111/S112 to be the determining factor in the enhancement of the transport. Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement. The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate. Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK-II sites at a distance of approximately 10-30 amino acid residues from the NLS. CK-II has been previously implicated in the transmission of growth signals to the nucleus. Our results suggest that CK-II may exert this role by controlling the rate of nuclear protein transport.

Amino Acid Sequence↗
PubMed1991

Salmonella contamination of poultry flocks in The Netherlands.

The contamination of poultry in the Netherlands with Salmonella enteritidis was tested. For this, different methods (detection of S. enteritidis in faecal samples of 25 g; detection of S. enteritidis in cloacal swabs; detection of S. enteritidis by serological testing of antibodies in serum) were compared for their efficiency to detect S. enteritidis in flocks of poultry. Testing of faecal samples clearly yielded the best results. This method was used in a transmission study, in which 14 flocks descending from a contaminated primary mother flock were screened for the presence of S. enteritidis. The method was also used for screening 49 flocks of laying hens and 52 flocks of broiler chickens throughout the Netherlands. From the transmission study it became clear that S. enteritidis, phage type 2 (Dutch phage set) was isolated both from the mother flock and from five of the descendent flocks. Screening of poultry flocks for the presence of salmonella revealed that salmonella was present in 47% of the layer flocks and in 94% of the broiler flocks. S. enteritidis was isolated from 15% of the flocks screened.

Animals↗
PubMed1991

Vasopressin V2-receptor mobile fraction and ligand-dependent adenylate cyclase activity are directly correlated in LLC-PK1 renal epithelial cells.

The role of hormone receptor lateral mobility in signal transduction was studied using a cellular system in which the receptor mobile fraction could be reversibly modulated to largely varying extents. The G-protein-coupled vasopressin V2-type receptor was labeled in LLC-PK1 renal epithelial cells using a fluorescent analogue of vasopressin, and receptor lateral mobility measured using fluorescence microphotolysis (fluorescence photobleaching recovery). The receptor mobile fraction (f) was approximately 0.9 at 37 degrees C and less than 0.1 at 10 degrees C, in accordance with previous studies. When cells were incubated for 1 h at 4 degrees C without hormone, and then warmed up to 37 degrees C and labeled with the vasopressin analogue, f increased from approximately 0.4 to 0.8 over approximately 1 h. The apparent lateral diffusion coefficient was not markedly affected by temperature pretreatment. Studies with radiolabeled vasopressin indicated that temperature pretreatment influenced neither receptor number nor binding/internalization kinetics. F-actin staining revealed that temperature change resulted in reversible changes of cytoskeletal structure. The maximal rate of in vivo cAMP production at 37 degrees C in response to vasopressin, but not to forskolin (receptor-independent agonist), was also markedly influenced by preincubation of cells at 4 degrees C, thus paralleling the effects of temperature preincubation on f. A linear correlation between f and maximal cAMP production was observed, suggesting that the receptor mobile fraction is a key parameter in hormone signal transduction in vivo. We conclude that mobile receptors are required to activate G-proteins, and discuss the implications of this for signal transduction mechanisms.

Actins↗
PubMed1991

p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV-40 T antigen proteins.

The nuclear import of transcription regulatory proteins appears to be used by the cell to trigger transitions in cell cycle, morphogenesis, and transformation. We have previously observed that the rate at which SV-40 T antigen fusion proteins containing a functional nuclear localization sequence (NLS; residues 126-132) are imported into the nucleus is enhanced in the presence of the casein kinase II (CK-II) site S111/112. In this study purified p34cdc2 kinase was used to phosphorylate T antigen proteins specifically at T124 and kinetic measurements at the single-cell level performed to assess its effect on nuclear protein import. T124 phosphorylation, which could be functionally simulated by a T-to-D124 substitution, was found to reduce the maximal extent of nuclear accumulation whilst negligibly affecting the import rate. The inhibition of nuclear import depended on the stoichiometry of phosphorylation. T124 and S111/112 could be phosphorylated independently of one another. Two alternative mechanisms were considered to explain the inhibition of nuclear import by T124 phosphorylation: inactivation of the NLS and cytoplasmic retention, respectively. Furthermore, we speculate that in vivo T124 phosphorylation may regulate the small but functionally significant amount of cytoplasmic SV-40 T antigen. A sequence comparison showed that many transcription regulatory proteins contain domains comprising potential CK-II-sites, cdc2-sites, and NLS. This raises the possibility that the three elements represent a functional unit regulating nuclear protein import.

Amino Acid Sequence↗