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M Zulauf

Publications and source records attributed to M Zulauf.

At least 19 recordsLinked to original sources
PubMed1995

Interferon gamma receptor extracellular domain expressed as IgG fusion protein in Chinese hamster ovary cells. Purification, biochemical characterization, and stoichiometry of binding.

Agents that antagonize the functions of interferon gamma (IFN gamma) are potential pharmaceuticals against several immunological and inflammatory disorders. IFN gamma receptor-immunoglobulin G fusion proteins (IFN gamma R-IgG) function as antagonists of endogenous IFN gamma and have longer half-lives in vivo in comparison with soluble IFN gamma receptors (sIFN gamma R), consisting of the extracellular region of the native sequence. A fusion protein comprising the extracellular domain of the human IFN gamma receptor and the hinge, CH2 and CH3 domains of the human IgG3 constant region, was expressed in Chinese hamster ovary cells. The IFN gamma R-IgG3 fusion protein was secreted into the culture medium as a 175-kDa glycoprotein and was purified over Protein G-Sepharose, DEAE-Sepharose, and size exclusion chromatography. IFN gamma R-IgG3 bound IFN gamma in solid phase assays and ligand blots, competed for the binding of radiolabeled IFN gamma to the cell surface receptor of Raji cells, and inhibited the IFN gamma-mediated antiviral activity with an efficiency at least one order of magnitude higher than that of the soluble receptor produced in the same expression system. Two IFN gamma R-IgG3 fusion proteins bound two IFN gamma dimers forming a complex of approximately 380 kDa. In immunodiffusion assays, the IFN gamma R-IgG3 fusion protein did not precipitate IFN gamma. Dissociation of bound IFN gamma from IFN gamma R-IgG3 was 2-fold slower than from the sIFN gamma R produced in insect cells.

Animals↗
PubMed1995

A 17-kDa CD4-binding glycoprotein present in human seminal plasma and in breast tumor cells.

We previously isolated gp17, a human seminal plasma glycoprotein, which specifically interacts with the D1-D2 region of CD4, a T cell surface molecule involved in antigen recognition mediated by helper T cells also acting as a receptor for the human immunodeficiency virus. In this study we report that monoclonal antibodies (mAb) reacting with gp17 are able to inhibit the binding of gp17 to immobilized soluble CD4. An immunohistochemical analysis shows that gp17 is also expressed in mammary tumor cells upon hormone treatment and in biopsies from breast cancer patients. A structural characterization of gp17, including amino acid sequencing, indicates that the protein has an extensive structural similarity with a glycoprotein designated as seminal actin-binding protein (SABP), also secreted by male sexual glands. SABP is in turn identical to gross cystic disease fluid protein-15 (GCDFP-15) or prolactin-inducible protein (PIP), a factor known as a highly specific and sensitive marker of primary and metastatic apocrine breast cancer. To establish further the correspondence of gp17 and GCDFP-15/PIP/SABP, the latter was expressed in bacteria from a cloned cDNA and purified by affinity chromatography to either anti-gp17 mAb-Sepharose or CD4-Sepharose. The purified recombinant protein is shown to inhibit the binding of labeled, pure g17 to immobilized soluble CD4. The finding that breast cancer cells express a protein able to interact with the CD4 domains involved in the recognition of class II major histocompatibility antigens suggests a possible mechanism by which a tumor may affect the activity of tumor-infiltrated CD4+ T cells.

Amino Acid Sequence↗
PubMed1994

The influence of patient reliability on visual field outcome.

The reliability of subjects to perform to perimetry correctly should be carefully evaluated to interpret visual field examinations adequately. Clinicians generally agree that numerous false-positive responses to catch trials cause measured thresholds to be falsely high and numerous false-negative responses cause measured thresholds to be falsely low. We studied the effect of false-positive and false-negative responses on the outcome of visual field measurements. Of 47 eyes, the results of 106 stable glaucomatous visual field tests (Program G1, Octopus 201, Interzeag, Schlieren, Switzerland) with false-positive responses and no more than one false-negative response to catch trials were compared to the results of reliable visual field tests (no false-positive and no more than one false-negative response) performed on the same eye. Similarly, 60 stable visual fields with false-negative responses and no more than one false-positive response were used to study the effect of false-negative responses on visual field sensitivities. Linear regression analysis disclosed a mean sensitivity increase of 1.5 dB for every 10% of false-positive responses (r = .34, P = .000) and a mean sensitivity decrease of 1.2 dB for every 10% of false-negative responses (r = .26, P = .04). These results may be used to help reduce the magnitude of unexplained long-term fluctuation in visual field interpretation.

Adult↗
PubMed1994

Normal visual fields measured with Octopus Program G1. I. Differential light sensitivity at individual test locations.

Exact knowledge of the normal variation of visual fields and the influence of age upon them is essential to differentiate normal from abnormal results, e.g., in glaucoma suspects. This study evaluates the normal variability of differential light sensitivity at individual test locations and incorporates a data base of a multicenter study performed with Octopus 201 perimeters using Program G1. The 824 fields thus obtained included 139 fields of 139 healthy volunteers who had undergone two previous visual fields and completed all three phases of the program. Sensitivity decreased linearly with age, an effect which for individual test locations ranged between 0.036 and 0.107 dB/year and which became more pronounced towards the periphery. On average, the age-related effects explained only 17% of the interindividual variation (range 4%-31%). The interindividual variation within a test averaged 4.6 dB2 and revealed a moderate, but quadratic increase towards the periphery. The intra-individual short-term variation was considerably lower, averaging 2.2 dB2. There was a similar modest, quadratic increase of the intra-individual variation towards the periphery. The results may help distinguish incipient disease manifestations from normal variation of perimetric results. Furthermore, the results give insight into the calculation of global visual field indices.

Adult↗
PubMed1994

Normal visual fields measured with Octopus-Program G1. II. Global visual field indices.

To complement results from individual test locations, clinicians also evaluate indices, as they give a succinct overview of the visual field. This, however, requires exact knowledge of their variability. The present study was designed to determine normal interindividual variability of global visual field indices and incorporates a data base of a multicenter study performed with Octopus 201 perimeters using Program G1. The 824 fields thus obtained included 139 fields of 139 healthy volunteers who had undergone two previous visual fields and completed all three phases of program G1. The index mean sensitivity showed a significant and linear decrease with increasing age, 0.064 dB/year of life. The indices loss variance, corrected loss variance, and short-term fluctuation did not correlate significantly with age. Percentiles are given for these visual field indices. While within the limits of normal values provided by the manufacturer for these indices, these results suggest that visual fields with "borderline" values require further clinical investigation in reliable, experienced subjects. The results may help clinicians to better evaluate global visual field indices and, therefore, to detect loss of visual function earlier.

Adult↗
PubMed1994

[Pupil perimetry with the Octopus 1-2-3. Initial experiences].

PURPOSE: Development of a clinically useful pupil perimetry on an OCTOPUS 1-2-3. MATERIALS AND METHODS: Various test parameters were investigated on a moderately modified OCTOPUS 1-2-3. We evaluated the pupillary responses of 122 eyes of 122 healthy volunteers qualitatively. RESULTS: Pleasing pupillary responses were obtained with the following parameters: 200 ms stimuli, Goldmann size 5 (1.5 degrees), background illumination 3 apostilb. Acoustic signals 0.5 seconds before stimulus presentation were helpful to minimize blinking artifacts. The pupillary reactions were fare stronger in central areas of the visual field than in mid-peripheral areas (i.e. 26 degrees). CONCLUSIONS: Pupil perimetry can be preformed on a moderately modified OCTOPUS 1-2-3. The results reveal considerable inter- and intra-individual variability. Equal pupillary responses at all eccentricities would improve subsequent mathematical evaluation.

Adult↗
PubMed1994

[Indications for stimulus 3 and 5 in automatic perimetry. Preliminary results].

PURPOSE: Goldmann stimulus size 3 and 5 were studied with automated perimetry in glaucoma patients to help develop specific guidelines for their use. METHODS: We examined 49 eyes of 49 glaucoma patients with both stimulus sizes 3 and 5 with Program G1 (all phases) of the Octopus 500 Perimeter. Fields were preformed sequentially, in random order, at the same sitting. RESULTS: The means of the indices for stimulus size 3 (+/- = standard deviation) were: Mean sensitivity 18.8 +/- 4.7 dB; loss variance 45.0 +/- 42.0 dB2; short-term fluctuation 2.1 +/- 0.45 dB. With stimulus size 5, mean sensitivity increased (Wilcoxon: p < 0.0005) by 8.1 +/- 1.7 dB, and short-term fluctuation decreased (Wilcoxon: p < 0.0005) by 0.3 +/- 0.6 dB. Scotomas measured with stimulus size 5 were shallower than with stimulus size 3, i.e., the pattern of visual field defects was better represented by stimulus size 3 in 13 eyes. Stimulus size 5 was preferred in 14 eyes because more information was available in the area of absolute scotomas. In 6 eyes, deep scotomas within 4 degrees of fixation were seriously underestimated with the larger stimulus. CONCLUSIONS: Stimulus size 5 should be used if more than 10% of test locations have absolute scotomas or the mean sensitivity falls below 15 dB with stimulus size 3. Complementary central fields with stimulus size 3 fields should be used when evaluation of the central field shows relative scotomas with either stimulus.

Aged↗
PubMed1993

An evaluation of clusters in the glaucomatous visual field.

We used a statistical cluster analysis to analyze patterns of loss in 76 visual fields with typical glaucomatous defects to identify natural groupings of test locations in the visual field. Eleven clusters in the Octopus Program G1 (Interzeag, Inc., Northboro, Massachusetts) visual field were thus defined. In a separate population of 70 early glaucomatous and 70 age-matched normal visual fields, the local mean defects within these clusters and the global mean defect were calculated to assess their relative abilities to discriminate between the two groups. The 11 clustered mean defects collectively had a sensitivity of 90% and a specificity of 93%; the global mean defect had a sensitivity of 81% and a specificity of 91%. Additionally, we examined the long-term fluctuation clusters of test locations compared to the long-term fluctuation of individual test locations. Four hundred ten visual fields of 93 clinically stable eyes of 67 patients with glaucoma, as well as 210 visual fields of 105 eyes of 105 normal subjects were studied. In the stable glaucoma group, mean fluctuation of clustered test locations was 3.5 dB2, and mean fluctuation of individual test locations was 7.0 dB2. In the normal group, the respective values were 0.6 dB2 and 1.8 dB2. Cluster analysis was effective in detecting localized loss and in dampening long-term fluctuation. We studied the use of clusters in distinguishing normal from glaucomatous as well as stable from deteriorating visual fields.

Adult↗
PubMed1993

Expression, purification and crystallization of fully active, glycosylated human interleukin-5.

Recombinant human interleukin-5 (hIL-5) has been expressed at high levels and produced in large quantities in baculovirus infected Sf9 insect cells. The glycosylated protein was purified using immuno-affinity chromatography and gel filtration. Purified hIL-5 has been crystallized using standard vapour diffusion techniques with PEG as a coprecipitant. The crystals belong to the C2 space group and diffract to 2 A.

Animals↗
PubMed1992

Stoichiometry of interaction between interferon gamma and its receptor.

The biological response of interferon gamma is mediated by binding to a specific cell-surface receptor. We investigated the stoichiometry of this binding using soluble receptors produced in prokaryotic and eukaryotic expression systems comprising the extracellular ligand-binding domain of the native protein. The ligand-receptor complexes were analyzed by cross-linking, chromatography, analytical ultracentrifugation and laser-light scattering. Cross-linking and chromatography showed that the stoichiometry of the interaction between ligand and receptor depends on the molar ratios of the two components mixed. All approaches confirmed that mixtures of ligand-receptor complexes are formed with one interferon-gamma dimer bound by one or two receptors. The soluble receptor produced in Escherichia coli mainly showed a ligand/receptor stoichiometry of 1:1, while the receptors produced in eukaryotic cells showed a stoichiometry of binding of 1:2. This apparent discrepancy is most likely due to the conformational heterogeneity of the Escherichia-coli-derived protein.

Amino Acid Sequence↗
PubMed1992

Long-term fluctuation of the visual field in glaucoma.

We examined 756 automated threshold visual fields of 167 eyes of patients with glaucoma to determine the magnitude of the long-term fluctuation at individual test locations. Eyes were grouped into clinically stable or nonstable groups according to predefined clinical criteria. At individual test locations with initial sensitivities of 25 to 30 dB in clinically stable eyes, the 5th to 95th percentile value for subsequent measurements was +/- 4 dB. For initial sensitivities of 20 dB, this 90% range was +/- 6 dB, and for sensitivities 15 dB or less, the 90% range of subsequent values spanned sensitivities from almost zero to normal values. Long-term fluctuation correlated with short-term fluctuation (r = .363, P less than .0005), but not with age or intraocular pressure. There was no correlation of long-term fluctuation with eccentricity after correcting for the decreased sensitivity associated with greater eccentricity. Long-term fluctuation was greater in the nonstable group than in the stable group (P less than .0005). These data provide limits for fluctuation in stable patients with glaucoma, which will help guide clinical decisions about visual field progression.

Adult↗
PubMed1992

2D crystallization: from art to science.

The techniques as well as the principles of the 2D crystallization of membrane and water-soluble proteins for electron crystallography are reviewed. First, the biophysics of the interactions between proteins, lipids and detergents is surveyed. Second, crystallization of membrane proteins in situ and by reconstitution methods is discussed, and the various factors involved are addressed. Third, we elaborate on the 2D crystallization of water-soluble proteins, both in solution and at interfaces, such as lipid monolayers, mica, carbon film or mercury surfaces. Finally, techniques and instrumentations that are required for 2D crystallization are described.

Aluminum Silicates↗
PubMed1992

Number of stimuli as a reliability parameter in perimetry.

Catch trials test patient performance during automated, static perimetry, but their adequacy to estimate reliability is uncertain even though up to 10% of the test time is reserved for catch trials. The 308 visual fields (program G1, all 3 phases, Octopus 201) of 308 eyes of 308 glaucoma, suspected glaucoma, and normal subjects were studied. The 108 visual fields (mean sensitivity > 10 dB; corrected loss variance < 50 dB2) without false responses to catch trials were considered reliable. A multiple linear regression analysis of these 108 fields was performed and revealed the following result (r2 = 0.751): Number of stimuli = 480 + (40.short-term fluctuation) + (8.8.the square root of the index corrected loss variance) - (2.2.mean sensitivity). This equation was used to estimate the number of stimuli required of a reliable subject to complete an examination. Excess stimuli would thus be a sign of reduced reliability. The difference between the estimated and the actual number of stimuli was called the 'stimulus discrepancy'. In 169 fields with false-positive and 58 fields with false-negative responses, the false-positive and false-negative responses correlated with the 'stimulus discrepancy' (r = 0.19, P = 0.014; r = 0.29, P < 0.026, respectively). The number of stimuli depends not only on reliability but also on the software and hardware of the perimeter. 'Stimulus discrepancy' may be an additional useful perimetric reliability parameter which does not require extra testing time.

Adult↗
PubMed1991

Density determination by analytical ultracentrifugation in a rapid dynamical gradient: application to lipid and detergent aggregates containing proteins.

A rapidly developing dynamical gradient can be formed in the analytical centrifuge when a buffer solution prepared in D2O is underlayered under the same buffer solution prepared in H2O in a specially designed double sector cell. In a short time the boundary layer spreads to form the gradient. Heavy particles (S greater than or equal to 10S) will band in the gradient corresponding to their density, which can be determined accurately. To this end, the buffer may contain density adjusting additives such as sucrose. We present results with this technique for lipid vesicles, for a Ca-antagonist bound to vesicles, as well as for a lipoprotein and reconstituted regular membrane protein-lipid arrays.

Animals↗
PubMed1991

Recombinant 55-kDa tumor necrosis factor (TNF) receptor. Stoichiometry of binding to TNF alpha and TNF beta and inhibition of TNF activity.

The extracellular domain of the 55-kDa TNF receptor (rsTNFR beta) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfr- cells. A chimeric fusion protein (rsTNFR beta-h gamma 3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG C gamma 3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected cell culture supernatants by TNF alpha- or protein G affinity chromatography and gel filtration. In a solid phase binding assay rsTNFR beta was found to bind TNF alpha with high affinity comparable with the membrane-bound full-length receptor. The affinity for TNF beta was slightly impaired. However, the bivalent rsTNFR beta-h gamma 3 fusion protein bound both ligands with a significantly higher affinity than monovalent rsTNFR beta reflecting most likely an increased avidity of the bivalent construct. A molecular mass of about 140 kDa for both rsTNFR beta.TNF alpha and rsTNFR beta.TNF beta complexes was determined in analytical ultracentrifugation studies strongly suggesting a stoichiometry of three rsTNFR beta molecules bound to one TNF alpha or TNF beta trimer. Sedimentation velocity and quasielastic light scattering measurements indicated an extended structure for rsTNFR beta and its TNF alpha and TNF beta complexes. Multiple receptor binding sites on TNF alpha trimers could also be demonstrated by a TNF alpha-induced agglutination of Latex beads coated with the rsTNFR beta-h gamma 3 fusion protein. Both rsTNFR beta and rsTNFR beta-h gamma 3 were found to inhibit binding of TNF alpha and TNF beta to native 55- and 75-kDa TNF receptors and to prevent TNF alpha and TNF beta bioactivity in a cellular cytotoxicity assay. Concentrations of rsTNFR beta-h gamma 3 equimolar to TNF alpha were sufficient to neutralize TNF activity almost completely, whereas a 10-100-fold excess of rsTNFR beta was needed for similar inhibitory effects. In view of their potent TNF antagonizing activity, recombinant soluble TNF receptor fragments might be useful as therapeutic agents in TNF-mediated disorders.

Animals↗
PubMed1991

Efficient purification of recombinant human tumor necrosis factor beta from Escherichia coli yields biologically active protein with a trimeric structure that binds to both tumor necrosis factor receptors.

A fast and efficient method for medium scale purification of recombinant human tumor necrosis factor beta (rTNF-beta) from Escherichia coli cells is described. The purified rTNF-beta displayed biological activity similar to rTNF-alpha in a WEHI 164 cell cytotoxicity assay. The titration curve of rTNF-beta and elution profiles of rTNF-beta in gel filtration experiments were different from those of rTNF-alpha. However, light scattering and ultra-centrifugation studies showed that both cytokines have trimeric structures in solution at 0.5 mg/ml, with minor differences in the distribution of nontrimeric species. rTNF-beta bound to purified 55- and 75-kDa TNF receptors with high affinity. The binding of rTNF-beta to either receptor was analyzed on Scatchard plots and compared with that of rTNF-alpha.

Binding, Competitive↗