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Biomedical subjects

H Hauser

Publications and source records attributed to H Hauser.

At least 163 records · Page 9Linked to original sources
PubMed1990

Uptake of indium-111 in the liver of mice following administration of indium-111-DTPA-labeled monoclonal antibodies: influence of labeling parameters, physiologic parameters, and antibody dose.

Liver uptake of indium-111 (111In) in mice was investigated following administration of 111In-DTPA murine monoclonal antibodies (111In-DTPA-MAbs) labeled by the cyclic anhydride method. Biodistribution of HPLC-purified 111In-DTPA-MAb preparations was checked with a low (0.2 micrograms) and a high (8.0 micrograms) MAb dose. Using Bio Gel P-30 for desalting the MAb-conjugates, 111In uptake in the liver amounted to 8%-9% of the injected dose (ID) and was independent from the MAb dose, the DTPA-to-MAb molar ratio, tumor growth and biologic variability (different MAbs and different strains of mice). Using Sephadex G-25 for desalting, 0.2 micrograms doses from 7 out of 26 preparations showed increased liver accumulation of 111In in non-tumor mice ranging from 15%-25% of ID. Corresponding high doses led to a "normal" value of 8%-9%. Increased liver uptake of the low dose could not be reduced by coadministration of the unconjugated MAb, but was normal after reinjection of "in vivo filtered" material. An inverse intracellular distribution of 111In activity between sediment and supernatant of liver homogenates, following the administration of the low and the high MAb dose, indicated an artifact of the labeling procedure rather than an inherent biological property of labeled MAbs.

Animals↗
PubMed1989

Large-scale preparation of recombinant platelet-derived growth factor AA secreted from recombinant baby hamster kidney cells.

The short isoform of platelet-derived growth factor A (PDGF-A) was expressed in a mammalian host (BHK-21 cell). A cell line was obtained that secreted up to 0.3 micrograms/10(6) cells recombinant PDGF-A chain homodimer/day into the medium. For large-scale production of supernatant, cells were grown either in roller bottles or in 2.5-1 stirred tank fermenters. A simple two-step procedure was developed to purify recombinant PDGF-AA (rPDGF-AA). The first step was adsorption onto porous glass and the final step was reversed-phase high-performance liquid chromatography. The yield was 0.2 mg/l supernatant. A total amount of 20-30 mg pure rPDGF-AA may be obtained from a single fermenter run. Sequence analysis showed the correct amino terminus and no internal proteolytic cleavages. The specific activity was 5 ng/ml for mouse AKR-2B cells. [125I]rPDGF-AA had an affinity constant of approximately 0.5 nM to these cells and 25,000 binding sites were estimated/cell.

Animals↗
PubMed1989

Expression of human interleukin-2 in recombinant baby hamster kidney, Ltk-, and Chinese hamster ovary cells. Structure of O-linked carbohydrate chains and their location within the polypeptide.

The similarity or identity of O-glycosylation in glycoproteins from natural sources or produced in heterologous cell lines, a central problem for the development of many biotechnologically relevant production processes, was examined using interleukin-2 (IL-2) as a model. Human interleukin-2 was constitutively expressed in several mammalian cell lines in high amounts. The recombinant proteins were purified to homogeneity and their carbohydrate structures were analyzed. Only the NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GalNAc oligosaccharide structure or the NeuAc alpha 2-3Gal beta 1-3GalNAc were found in all IL-2 preparations secreted from recombinant Ltk-, Chinese hamster ovary, and baby hamster kidney cell lines. The O-linked chains were exclusively linked to Thr in position 3 of the polypeptide chain which is the carbohydrate attachment site in natural human IL-2. The proportions of O-glycosylated versus nonglycosylated forms of the protein secreted by each recombinant cell line were independent of productivity or of cell culture conditions. Our results show that O-glycosylated human IL-2 can be produced by applying recombinant DNA technology in heterologous cell lines with the same type of post-translational modification that is observed for the protein secreted from natural T lymphocytes.

Animals↗
PubMed1989

Infrared and 31P-NMR studies of the interaction of Mg2+ with phosphatidylserines: effect of hydrocarbon chain unsaturation.

Infrared and 31P-NMR spectra of aqueous dispersions of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and ox brain phosphatidylserine in the presence of excess Mg2+ have been recorded. A consistent picture emerges from the application of infrared and 31P-NMR spectroscopy to Mg2+-PS interactions. Mg2+ forms crystalline complexes with saturated phosphatidylserines, such as DMPS, and probably with POPS. In these crystalline PS-Mg2+ complexes the phosphate group loses its water of hydration but the serine carboxylate remains hydrated. Furthermore, there is formation of an additional hydrogen bond to one of the ester carbonyl groups of DMPS, and interchain interactions appear to be enhanced as reflected by a tighter packing of the fatty acyl chains. One main conclusion of this work is that Mg2+ binding to PS bilayers shows a gradation, the binding is in the order DMPS greater than POPS greater than ox brain PS greater than DOPS. The molecular area increases in the order DMPS less than ox brain PS less than POPS less than DOPS and is apparently an important parameter determining the affinity of PS for Mg2+. The general trend is that with increasing molecular area, and hence spacing of the ligands, the binding of Mg2+ decreases. While PS with two saturated fatty acyl chains forms tightly packed, crystalline Mg2+ complexes with an immobilized headgroup, the unsaturated PS molecules such as ox brain PS and DOPS interact only weakly with Mg2+. Their interaction seems to be restricted to electrostatic shielding, since no major changes in molecular conformation, chain packing and headgroup hydration are found. The interaction of POPS with Mg2+ is intermediate between that of saturated PS and that of DOPS. POPS exhibits a higher affinity for Mg2+ than ox brain PS, although their molecular areas (and the surface charge density) are approximately the same. This apparent anomaly is proposed to be due to a discreteness of charge effect. It is proposed that a lipid surface with regularly spaced polar groups has a higher affinity for binding Mg2+.

Calcium↗
PubMed1989

A new electron spin resonance assay for membrane asymmetry and entrapped volume of unilamellar lipid vesicles based on photoreduced flavin adenine dinucleotide.

A new ESR assay has been developed for the characterization of unilamellar lipid vesicles. It is based on the reduction by photogenerated FADH2 of amphiphilic spin-labels having the spin in the polar group. FADH2 is generated in situ under anaerobic conditions from its oxidized form (FAD) by photoreduction in the presence of excess EDTA as the reducing agent. Photoreduction is induced by exposing the FAD/EDTA mixture to white light of a commercial slide projector. FADH2 as an impermeable agent reduces spin-label molecules located on the outer layer of the bilayer that are readily accessible in a first fast reaction; spin-label located on the inner layer of the bilayer is reduced in a second slow reaction. The ESR assay is suitable for the routine characterization of unilamellar membrane vesicles: it allows the determination of the vesicle size, the entrapped volume, the bilayer asymmetry, the bilayer integrity, and the vesicle stability. The ESR assay developed is of general applicability: it can be used with charged and uncharged bilayers which may be labeled with either neutral or charged spin-labels. An assessment of the new ESR assay is given in comparison to the existing ascorbate method which uses sodium ascorbate as the reducing agent. Various other potential reducing agents for spin-labels have been tested and found unsuitable for the ESR assays discussed here.

Ascorbic Acid↗
PubMed1989

Interactions of metal ions with phosphatidylserine bilayer membranes: effect of hydrocarbon chain unsaturation.

A combination of surface monolayer, scanning calorimetry, 31P NMR, and spin-label ESR techniques has been used to monitor the interactions of monovalent (NH4+, Na+, and Li+) and divalent (Ca2+) cations with phosphatidylserines (PS) differing in their levels of chain unsaturation. Comparisons are made between the disaturated dimyristoyl-, dipalmitoyl-, and dihexadecyl-PS (DMPS, DPPS, and DHPS), saturated cis-monounsaturated palmitoyloleoyl-PS (POPS) (and bovine brain PS), di-trans-monounsaturated dielaidoyl-PS (DEPS), and di-cis-monounsaturated dioleoyl-PS (DOPS). Na+ and NH4+ cations interact weakly with all PS monolayers and bilayers without significant changes in molecular conformation, chain packing, or headgroup dynamics and without dependence on chain composition. In contrast, considering these structural and dynamic parameters, Li+ shows a gradation in its interaction with PS (DMPS greater than POPS approximately bovine brain PS greater than DOPS), suggesting that Li+-PS interactions depend on the interfacial properties of the PS molecules (e.g., surface area). Finally, Ca2+ interacts strongly with all PS monolayers and bilayers, without obvious chain selectivity. Thus, ion binding to PS depends not only on the properties of the cation (Na+ vs Li+ vs Ca2+) but also on the molecular details of the PS membrane surface.

Calcium↗
PubMed1989

Phase behaviour of mixtures of lipid X with phosphatidylcholine and phosphatidylethanolamine.

The effect of increasing concentrations of lipid X (2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine 1-phosphate) on the phase behaviour of EPC (egg phosphatidylcholine) and EPE (egg phosphatidylethanolamine) is studied at a pH greater than or equal to 7 where lipid X carries one to two negative charges. Small amounts of lipid X (molar ratio approximately 0.01) induce continuous swelling of EPC and EPE bilayers and consequently the formation of large unilamellar vesicles in excess water. In many respects, the effect of lipid X on EPC and EPE bilayers is similar to that of phosphatidic acid. However, lipid X/EPC mixtures form micelles in excess lipid X whereas mixtures of phosphatidic acid/EPC vesiculate at all ratios. The same is true for lipid X/EPE mixtures. Small unilamellar vesicles of an average diameter of 40 nm form spontaneously upon dispersion of a dry lipid X/EPE film (molar ratio = 10). Unsonicated dispersions of lipid X/EPC (molar ratio = 1) are subjected to pH-jump treatment which involves raising of the pH to 11-12 and subsequent lowering of the pH to between 7.5 and 8.5. Such a treatment has little effect on the vesicle size and size distribution as compared to a control dispersion at pH 8.2. The mean size is determined to be 92 +/- 60 nm. Electron micrographs of freeze-fractured samples of lipid X/EPC (molar ratio = 1) reveal the presence of mainly micelles at pH 12. Upon lowering the pH to neutrality these micelles become unstable and aggregate/fuse rapidly to unilamellar vesicles (average diameter 95 +/- 40 nm). Sonication of equimolar mixtures of lipid X and EPC at pH 7 yields small unilamellar vesicles of a diameter of 20-25 nm as well as mixed micelles of a size between 15 and 17 nm. This behaviour is again different from that of mixed EPC/phosphatidic acid dispersions which form small unilamellar vesicles. The presence of lipid X in such mixtures does not prevent the aggregation/fusion to larger vesicles during freezing of the dispersion. As with pure EPC bilayers, stabilization is, however, achieved in the presence of 10% sucrose. This indicates that the covalently bonded glucosamine group of lipid X cannot substitute water of hydration in neighbouring EPC molecules.

Chromatography, Gel↗
PubMed1989

Synthesis and localization of myeloperoxidase protein in transfected BHK cells.

Processing and localization of myeloperoxidase was studied in nonmyeloid cells. For this purpose BHK cells were transfected with human myeloperoxidase cDNA. In the transfected cells a protein with mol wt of 85,000 was found, which reacted with the specific anti-human myeloperoxidase antiserum. In size and in sensitivity to endo-beta-N-acetylglucosaminidase H this protein resembled the myeloperoxidase precursor synthesized in human promyelocytes. Unlike in the promyelocytes, in BHK cells the 85,000-Da protein was not converted to 60,000- and 14,000-Da polypeptides of the mature enzyme. In Percoll gradients the protein was found predominantly in the light membrane fractions. Microscopic examination revealed a conspicuous immune reaction over the endoplasmic reticulum and nuclear membranes and a moderate labeling over lysosome-like organelles. Pulse-chase experiments indicated that the protein was slowly released from the endoplasmic reticulum; after 1 day the protein was found in similar amounts in cells and in the medium. The secreted protein contained at least one endo-beta-N-acetylglucosaminidase-resistant oligosaccharide. It is suggested that normal intracellular segregation of myeloperoxidase depends on a signal or component, which is not or incompletely expressed in BHK cells.

Animals↗
PubMed1989

Mechanism of spontaneous vesiculation.

Both naturally occurring and synthetic phosphatidic acid (PtdOH) molecules show the phenomenon of spontaneous vesiculation on jump in pH value. This method involves a transient increase in pH of smectic PtdOH dispersions to values between 10 and 12. Such a pH increase induces spontaneous vesiculation with the formation of small unilamellar vesicles of diameter less than 50 nm as shown by 31P NMR. Both high-resolution and broad-line 31P NMR were used to study the mechanism of this process. When the pH of unsonicated PtdOH dispersions is raised to pH 10-12, lipid molecules on the outer monolayer of the bilayer become fully ionized. The second pK value of PtdOH in bilayers is 8.6 +/- 0.3, determined by 31P NMR. PtdOH molecules on the inner monolayer remain partially protonated. 31P NMR provides unambiguous evidence that the "pH-jump" treatment produces a pH gradient across the PtdOH bilayer. The orientation of the pH gradient is such that the pH in the external medium is 3-5 pH units higher than the internal pH. Associated with the pH gradient is a transverse packing asymmetry: partially protonated PtdOH molecules in the inner layer of the bilayer are more tightly packed than fully ionized molecules present in the outer layer. The pH gradient generated by the pH jump is proposed as the energy source that drives the spontaneous formation of highly curved vesicles.

Hydrogen-Ion Concentration↗
PubMed1989

Isolation and functional characterization of the murine interferon-beta 1 promoter.

A murine cosmid clone harboring the single-copy interferon-beta 1 (IFN-beta 1) gene and extended flanking sequences was isolated. The functional IFN-beta 1 promoter is contained within a 170-bp DNA fragment located 5' of the coding sequence. This was shown by fusion of this fragment to a heterologous reporter gene and transient as well as stable expression in mouse L and monkey CV-1 cells. With the help of these functional assays, it could be demonstrated that the 5'-flanking sequences are the target for the typical regulatory action of common type I IFN activators. DNA sequencing reveals a considerable homology to the human IFN-beta 1 promoter within the 280 upstream base pairs. The homology is particularly pronounced within the DNA region containing the virus responsive element (VRE). This phenomenon may explain the similarity of both genes in the mode of regulation. The mouse promoter fragment compared with the human equivalent was shown to be several times more efficient in transcriptional activation in murine and primate cells.

Animals↗
PubMed1989

Acute pancreatitis and normoamylasemia. Not an uncommon combination.

A consecutive series of 352 attacks of acute pancreatitis (AP) was studied prospectively in 318 patients. AP was ascertained by contrast-enhanced CT scan in all but four cases in which diagnosis was made at operation or autopsy. Sixty-seven of these cases (19%) had normal serum amylase levels on admission (i.e., less than 160 IU/L, a limit that includes 99% of control values), a figure considerably higher than generally admitted. When compared to AP with elevated serum amylase, normoamylasemic pancreatitis was characterized by the following: (1) the prevalence of alcoholic etiology (58% vs. 33%, respectively, p less than 0.01), (2) a greater number of previous attacks in alcoholic pancreatitis (0.7 vs. 0.4, p less than 0.01); and (3) a longer duration of symptoms before admission (2.4 vs. 1.5 days, p less than 0.005). In contrast AP did not appear to differ significantly in terms of CT findings, Ranson's score, and clinical course, when comparing normo- and hyperamylasemic patients, although there was a tendency for normoamylasemic patients to follow milder courses. Serum lipase was measured in 65 of these normoamylasemic cases and was found to be elevated in 44 (68%), thus increasing diagnostic sensitivity from 81% when amylase alone is used to 94% for both enzymes. A peritoneal tab was obtained in 44 cases: amylase concentration in the first liter of dialysate was greater than 160 IU/L in 24 cases (55%), and lipase was greater than 250 U/L in 31 cases (70%). Twelve of these 44 cases had low peritoneal fluid and plasma concentrations for both enzymes. Thus little gain in diagnostic sensitivity was obtained when adding peritoneal values (96%) to serum determinations. AP is not invariably associated with elevated serum amylase. Multiple factors may contribute to the absence of hyperamylasemia on admission, including a return to normal enzyme levels before hospitalization or the inability of inflamed pancreases to produce amylase. Approximately two thirds of cases with normal amylasemia were properly identified by serum lipase determinations. AP does not appear to behave differently when serum amylase is normal or elevated, and should therefore be submitted to similar therapeutic regimens in both conditions.

Acute Disease↗
PubMed1988

Preferred conformation and dynamics of the glycerol backbone in phospholipids. An NMR and X-ray single-crystal analysis.

The conformation of the glycerol group of a number of diacyl and monoacyl (lyso) phospholipids differing in the chemical nature of the head group was studied by 1H high-resolution NMR and X-ray crystallography. The NMR measurements were carried out with solutions or micellar dispersions of the lipids in deuteriated organic solvents or 2H2O. Both solutions, in which the lipid is present as monomers, and lipid micelles give rise to good high-resolution NMR spectra exhibiting spin coupling hyperfine interactions. From 1H spin coupling it is concluded that there are two stable conformations about the glycerol C(2)-C(3) bond of phospholipids. One of these (rotamer A) is characterized by torsion angles theta 3 = antiperiplanar, theta 4 = +synclinal, and the other (rotamer B) by theta 3 = +synclinal, theta 4 = -synclinal. In both rotamers A and B the ester oxygens on the glycerol carbon atoms C(2) and C(3) are synclinal, and hence both types of rotamers readily allow the parallel alignment of the two hydrocarbon chains. By comparison of NMR and single-crystal X-ray data it is obvious that both conformations are minimum free energy conformations. Rotamer A is the conformation prevailing in phospholipid single-crystal structures. The conformation of rotamer B is also found in phospholipid single-crystal structures though to a lesser extent, e.g., in 2,3-dilauroyl-DL-glycero-1-phospho-N,N-dimethylethanolamine and 2,3-dimyristoyl-D-glycero-phospho-DL-glycerol. NMR measurements indicate that in liquid crystals the diacylglycerol part of phospholipids fluctuates between the two stable staggered conformations of rotamers A and B.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycerol↗
PubMed1988

Isolation of overproducing recombinant mammalian cell lines by a fast and simple selection procedure.

The expression of genes transfected into mammalian cells is critically dependent on both the copy number and the site of integration. This conclusion was derived from the transfection of a plasmid containing three linked genes, which guarantees the same dose of each gene in the recipient cell clones. The selection pressure imposed by the usual concentrations of the neomycin analogue G418 or puromycin alone is low and yields cell clones with widely differing transcription rates. Time-consuming screening procedures, which are generally applied to obtain high-yield cell lines, can be obviated by the simultaneous transfer of two drug-resistance genes (neo plus dhfr, or neo plus pac). By a combined selection procedure only cells with high-level expression for the first marker will survive upon exposure to increasing concentrations of the second antibiotic. Generally, these cells also exhibit high expression levels for the non-selected gene. Many of the selected clones exhibit increased copy numbers of the respective gene but some of them owe their high productivity to a favorable position of only one or a few copies of the gene. Screening for high-yield clones using the combined selection protocol introduced here provides rapid and simple access to authentic and mutant gene products.

Animals↗
PubMed1988

Vectors for efficient expression in mammalian fibroblastoid, myeloid and lymphoid cells via transfection or infection.

We have constructed two related types of multi-cloning mammalian expression vectors. The first, pMPSVEH/HE, carries the promoter of the myeloproliferative sarcoma virus (MPSV). This promoter was found to be stronger than both the SV40 early and the trans-activated human immunodeficiency virus promoters in many cell lines including human and rodent fibroblastoid, lymphoid or myeloid cells. The other, pBEH/HE, carries the simian virus 40 (SV40) early promoter and origin of replication. This offers the possibility of encapsidation in SV40 pseudovirions and subsequent gene transfer into, e.g., hemopoietic cells, via infection. The usefulness of the expression systems was tested with a number of genes and cell lines.

Animals↗
PubMed1988

Interaction of hemoglobin derivatives with liposomes. Membrane cholesterol protects against the changes of hemoglobin.

Hemoglobin (Hb) was incubated with small unilamellar vesicles of different lipid compositions in physiological or low pH-low ionic strength media, and the alterations in the protein and bilayer structure were analyzed by measuring (i) the sedimentation properties and elution pattern of the vesicles upon gel filtration, (ii) the quenching effect of Hb on the fluorescence of membrane-embedded probes, (iii) the intrinsic fluorescence of Hb, and (iv) the Soret spectra of Hb. The results indicate complex formation between Hb and the membrane, followed by intercalation of the protein into the bilayer. These processes can lead to secondary alterations, including aggregation, peroxidative decomposition of unsaturated fatty acids, unfolding of Hb, oxidation of the heme iron, displacement of the heme relative to globin, and oxidative or nonoxidative deconjugation of the porphyrin ring. Complex formation and intercalation of Hb into the bilayer are primarily due to hydrophobic interaction between the protein and the membrane, whereas displacement of the heme and its nonoxidative deconjugation are elicited by ionic interaction between the heme and negative membrane surface charges. These charges, on the other hand, inhibit the oxidative processes in unsaturated lipid vesicles. The rate and extent of alterations both in the protein and in the membrane show inverse correlation with the stability of the heme-globin linkage. The liquid-crystalline phase state promotes the penetration of Hb into the bilayer, whereas displacement of the heme is most expressed in the case of saturated bilayers in the gel state. Cholesterol inhibits both the hydrophobic and ionic interactions between the protein and bilayer, most remarkably the partition of the heme into negatively charged saturated bilayers. It is suggested that the effects of cholesterol are mediated through changes in membrane fluidity.

Animals↗
PubMed1988

[Classification of colorectal cancers: current results from the catchment area of the Graz Institute of Pathology].

In comparison with the results of Dukes the percentage of stage Dukes A of colorectal carcinomas could be increased from 15% to about 27%, whereas the percentage of stage Dukes C decreased from 51 to 40% in some international medical centers during the seventies of this century. Our results in Styria are comparable. However, there was no further progress in the early diagnosis of colorectal carcinomas in Styria from 1984 to 1986 (42% of Dukes C stage cases) as compared with 1974 to 1979 (41% of Dukes C stage cases). The percentage of endoscopically removed tumors increased from 3% (1974 bis 1979) to 9% (1984 to 1986) of all curatively treated carcinomas. Because of the fact that the endoscopically removed tumors (stage pT1pNx) could not be classified according to Dukes, the percentage of Dukes stage A decreased from 30% (1974 to 1979) to 24% (1984 to 1986). But the small numbers of endoscopically removed tumors did not significantly influence the percentage of Dukes C stages. There was only partial success in the predominantly rural areas in which the percentage of Dukes C stages decreased from 47% (1974 to 1979) to 39% (1984 to 1986). 39% Dukes stage C in rural areas correspond to the percentage of the predominantly municipal areas (40%). This progress can be explained by the availability of colonoscopy to all hospitals in 1984 to 1986, which was not the case previously.(ABSTRACT TRUNCATED AT 250 WORDS)

Austria↗