PubMed Health⌕ Search

Biomedical subjects

H Hauser

Publications and source records attributed to H Hauser.

At least 109 records · Page 6Linked to original sources
PubMed1994

CD4 expressing human 293 cells as a tool for studies in HIV-1 replication: the efficiency of translational frameshifting is not altered by HIV-1 infection.

An adherent human cell line (293) was made susceptible for HIV-1 infection by transfer of a CD4 expression plasmid. These cells could be infected with HIV-1 and produced infectious virus up to a titer of 10(6) TCID50/ml releasing p24 protein up to 1 micrograms/ml. Since they can be efficiently transfected with reporter genes, these cells are a suitable model system to monitor biochemical events during productive infection of HIV-1 and can be used for antiviral drugs. Translational frameshifting determines the balance of the structural Gag versus the catalytic Pol proteins which is probably crucial for correct virus assembly. We have genetically engineered CD4 expressing 293 cells with a sensitive in vivo reporter system to monitor the extent of frameshifting in HIV-1-infected versus uninfected cells. During the time course of productive HIV-1 infection the low efficiency of ribosomal frameshifting is not altered.

CD4 Antigens↗
PubMed1994

Short-chain phosphatidylcholines as superior detergents in solubilizing membrane proteins and preserving biological activity.

The solubilization of plasma and organelle membranes by diheptanoylphosphatidylcholine (DHPC) has been studied. This short-chain phosphatidylcholine is shown to act as a mild detergent, solubilizing effectively both kinds of membranes at DHPC concentrations of 10-20 mM (0.5-1%). The size of the resulting mixed protein-lipid-DHPC micelles ranges between 5 and 8 nm. The protein conformation and hence the enzymatic activity are well preserved over a rather large DHPC concentration range (up to 4-5 times the DHPC concentration required for solubilizing the membranes). Evidence is presented that short-chain phosphatidylcholines are superior to most detergents commonly used by biochemists. This is true not only regarding its excellent dispersing power on both phospholipid bilayers (Gabriel & Roberts, 1986) and biological membranes but also as to its capacity to preserve the native protein structure and hence enzymatic activity in the solubilized state. Due to its special properties DHPC lends itself very well not only to membrane solubilization but also to the purification of the solubilized membrane proteins and reconstitution of the proteins into simple lipid bilayers. Concerning the mechanism of membrane solubilization, evidence indicates that DHPC interacts primarily with the lipid bilayer of the membrane and not with the membrane proteins. DHPC solubilizes membranes by being distributed into the lipid bilayer and breaking it up. In the resulting small mixed micelles, the protein remains associated with its preferred intrinsic membrane lipids and is thus stabilized. The protein-intrinsic lipid complex is successfully shielded from unfavorable contacts with H2O by DHPC-intrinsic lipid interactions.

Bacteriochlorophylls↗
PubMed1994

Absorption of monoacylglycerols by small intestinal brush border membrane.

The absorption of monoacylglycerol by small intestinal brush border membrane is a passive process, i.e., the movement of monoacylglycerol from small unilamellar phospholipid vesicles as donor particles through the aqueous medium and the incorporation into the outer monolayer of the lipid bilayer of the brush border membrane are passive processes involving diffusion of the lipid along a concentration gradient. Small unilamellar vesicles of egg phosphatidylcholine containing 1 mol% of radiolabeled hexadecylglycerol were used as donor, and rabbit small intestinal brush border membrane vesicles or intact enterocytes isolated from pig jejunum, as acceptor. Hexadecylglycerol was employed as a lipase-resistant model compound for monoacylglycerols. Both acceptor membranes behave similarly in terms of hexadecylglycerol absorption: the kinetics of hexadecylglycerol absorption are biphasic. The initial fast phase is due to the movement of hexadecylglycerol from the donor particle through the aqueous medium to the outer lipid monolayer of the acceptor membrane, and the second slow phase probably involves the flip-flop motion of hexadecylglycerol from the outer to the inner monolayer of the acceptor membrane. The values for the pseudo-first-order rate constants of the initial fast phase for hexadecylglycerol absorption are relatively large and primarily determined by the high solubility (cmc) of hexadecylglycerol in aqueous media. The pseudo-first-order rate constants depend linearly on the protein (lipid) concentration of the acceptor membrane, indicating that the on rate of the hexadecylglycerol into the brush border membrane is rate limiting. The mechanism of the hexadecylglycerol absorption involves mainly monomer diffusion and probably collision-induced transfer.

Animals↗
PubMed1994

Probing conformational states of spin-labeled aspartate aminotransferase by ESR.

Mitochondrial aspartate aminotransferase was selectively labeled with various maleimide-linked nitroxide spin labels at the conformationally sensitive Cys166. The mobility of the spin group was found to increase with increasing length of the spacer between the nitroxide and maleimide moiety. The label with the ethylcarbamoyl group, a spacer of intermediate length, responded sensitively to conformational changes of aspartate aminotransferase. The modification with this label decreased the enzymic activity to 30% of its initial value and increased the affinity for various substrates and inhibitors 5-10-fold. Identical ESR spectra were obtained for the pyridoxal and pyridoxamine form of the enzyme. These spectra are complex, consisting of an isotropic and at least two anisotropic components. The spectral complexity is attributed to different modes of interaction of the spin label with its local protein environment giving rise to different motional states. The same changes in the ESR spectra have been observed upon formation of the adsorption complex of the pyridoxal form with a competitive inhibitor and on formation of covalent intermediates of the transamination reaction. Essentially, the isotropic component is converted to a new anisotropic one as the local environment changes due to a conformational adaptation of aspartate aminotransferase. The ESR data are consistent with an equilibrium between two conformational states of the enzyme but inconsistent with individual protein conformations of the various intermediates of the transamination reaction. The two conformational states may be assigned to the open and closed conformations as defined by X-ray crystallography. In the adsorption complex of the pyridoxal enzyme, and in the covalent intermediates, the two-state equilibrium appears to be shifted towards the closed conformation in which the spin label is more rigidly bound, as also suggested by molecular dynamic simulations of the label modelled into aspartate aminotransferase. In contrast the formation of adsorption complexes between the pyridoxamine form and aspartate or maleate was not accompanied by the same shift of the conformational equilibrium.

Aspartate Aminotransferases↗
PubMed1994

Conformation of phosphatidylserine in bilayers as studied by Fourier transform infrared spectroscopy.

The 13C labeled lipid 1[1'-13C]DPPS-NH4+ and its metal salts were used to unambiguously assign all carbonyl vibrations in the infrared spectrum of phosphatidylserines. It is shown that the C=O stretching band at 1741 cm-1 of phosphatidylserines previously assigned to the sn-1 C = O vibration contains contributions from both the sn-1 and the sn-2 carbonyls. The C=O stretching band at frequencies between 1715 and 1730 cm-1 previously assigned to the sn-2 C=O vibration also contains contributions from both carbonyl groups. The frequency dependence observed with the ester carbonyls primarily reflects hydrogen bonding and the polarity of the immediate vicinity. Conformational changes are accounted for in terms of frequency shifts if the conformational change involves the disposition of the C=O groups and in turn the hydrogen bonding properties. The infrared spectra of phospholipids dispersed in aqueous medium in the liquid crystalline state are inconsistent with a simple phospholipid conformation, e.g., with a conformation as found in the single-crystal structure of 1,2-dimyristoyl-sn-phosphatidylcholine and 1,2-dilauroyl-rac-phosphatidylethanolamine. The spectra support the hypothesis proposed earlier (Hauser et al., Biochemistry, 1988) on the basis of existing single-crystal phospholipid structures and NMR evidence. The hypothesis states that several conformations are present in liquid crystalline phospholipid dispersions.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Isotopes↗
PubMed1994

Expression of recombinant antibody and secreted alkaline phosphatase in mammalian cells. Influence of cell line and culture system upon production kinetics.

The growth and productivity of an Sp2/0 cell line, F3b10, expressing a recombinant antibody (rAb) and BHK21 cells expressing either the same rAb from the same plasmids (BHK.IgG) or secreted alkaline phosphatase (SEAP) (BHK.SEAP) were investigated. The F3b10 line was grown as a single cell suspension. The BHK lines were grown either as suspended natural aggregates or on Cytodex 3 microcarriers. The data for F3b10 showed that the cell-specific rAb production rate (QsrAb) increased in parallel with increases in the specific growth rate (mu). A similar result was obtained for suspended aggregate cultures of both recombinant BHK cell lines. In contrast, for microcarrier cultures of both BHK cell lines, Qsproduct increased as mu decreased. This report shows that the relationship between cell growth and Qsproduct for the cell lines and products studied is dependent upon the culture process. In systems where recombinant cells are growing as a single cell suspension or within a natural suspension aggregrate, Qsproduct increased with increases in mu. In such systems, the cells have a rounded morphology. When cells were grown on microcarriers. Qsproduct decreased as mu increased. Cells growing attached to a surface are flat and elongated. The observed differences in the relationship of Qsproduct to mu are correlated with changes in cell morphology. The relationship between Qsproduct and mu is also affected by the choice of cell line.

Alkaline Phosphatase↗
PubMed1994

Perforation of the cecum by a toothpick. Case report and review of the literature.

Perforation of the colon can have any number of causes. Most often, it is the result of carcinoma or diverticulitis; specific inflammatory disease of the large intestine is a less common cause. Other possibilities are iatrogenic perforation, perforation as a result of blunt or penetrating abdominal trauma, and ingestion of a foreign body. A case of perforation of the cecum by a toothpick 3 weeks after consumption of a beef olive is reported; possible diagnostic problems, other conditions that need to be considered in the differential diagnosis and treatment are discussed.

Aged↗
PubMed1994

Detection of mycoplasma contaminations by the polymerase chain reaction.

The polymerase chain reaction (PCR) has been used for the general detection of Mollicutes. 25 Mycoplasma and Acholeplasma species were detected including important contaminants of cell cultures such as M. orale, M. arginini, M. hyorhinis, M. fermentans, A. laidlawii and additional human and animal mycoplasmas. PCR reactions were performed using a set of nested primers defined from conserved regions of the 16S rRNA gene. The detection limit was determined to be 1 fg mycoplasma DNA, which is equivalent to 1-2 genome copies of the 16S rRNA coding region. The identity of the amplification products was confirmed by agarose gel electrophoresis and restriction enzyme analysis. DNA from closely and distantly related micro-organisms did not give rise to specific amplification products. The method presented here offers a much more sensitive, specific and rapid assay for the detection of mycoplasmas than the existing ones.

Base Sequence↗
PubMed1994

Screening retroviral packaging cells for highly efficient virus production by using a combined selection procedure.

To facilitate the screening for clones of transfected packaging cells producing a high yield of recombinant retrovirus, we present a fast and simple method for the isolation of overexpressing cells. By this method the efficiency of virus production can generally be enhanced 10- to 100-fold by application of high selection pressure. Cell lines which exhibit titers of up to 10(8) CFU/ml were obtained.

3T3 Cells↗
PubMed1994

The sequences of and distance between two cis-acting signals determine the efficiency of ribosomal frameshifting in human immunodeficiency virus type 1 and human T-cell leukemia virus type II in vivo.

We have analyzed in cell culture the sequence elements that control the level of ribosomal frameshifting in the human T-cell leukemia virus type II (HTLV-2) gag-pro junction. The slippery sequence of HTLV-2 is sufficient to dictate a basal level of frameshifting. This level is enhanced by its upstream sequence context and by the downstream stem-loop structure which is located at an optimal distance of 7 bases. Frameshifting in human immunodeficiency virus gag-pol is similar to that of HTLV-2 gag-pro. However, experiments using hybrid cassettes of HTLV-2 and human immunodeficiency virus type 1 frameshift elements show that while the slippery sequence of HTLV-2 is less efficient, the stem-loop structure is a more efficient enhancer.

Base Sequence↗
PubMed1993

Thermodynamic stability and osmotic sensitivity of small unilamellar phosphatidylcholine vesicles.

Evidence is presented to show that small unilamellar phosphatidylcholine vesicles with a diameter of approx. 20 nm are osmotically sensitive. Such vesicles respond to osmotic pressure by swelling or shrinking depending on the direction of the applied salt gradient. This is true for small unilamellar vesicles of egg phosphatidylcholine and dimyristoylphosphatidylcholine below and above their crystal-to-liquid crystal transition temperature. At the transition temperature the vesicles are osmotically insensitive due to the increased bilayer permeability resulting in rapid dissipation of salt gradients. Positive salt gradients produce shrinking and collapse of spherical phospholipid vesicles to disks. Shrinking of vesicles is associated with H2O and solute efflux, but only limited solute influx. Clustering of lipid molecules in the bilayers of the resulting disks can be detected by EPR spin labeling. Negative salt gradients produce swelling of vesicles which is associated with H2O and solute influx. Our experiments are consistent with an osmotically perturbed bilayer. In the presence of osmotic gradients the influx and efflux of H2O is coupled with the movement of ions and small molecules which in the absence of salt gradients or osmotic stress cannot pass the phospholipid bilayer. However, during osmotically induced shrinking and swelling of SUV the integrity of the phospholipid bilayer is maintained to the extent that vesicles do not break, and therefore equilibration between external medium and vesicle cavity does not take place.

Dimyristoylphosphatidylcholine↗
PubMed1993

Specificity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines.

The polymerase chain reaction (PCR) amplification was used for the detection of mycoplasma contamination in 42 continuous cell lines. Using the microbiological cultivation on agar as the reference method, 29 cell lines were regarded as positive and 13 cell lines as negative. The double-step PCR analysis employed nested primers that anneal to gene sequences coding for the evolutionarily conserved 16 S rRNA of some 25 different mycoplasma species (including the ones most commonly found in cell cultures). In terms of the positivity or negativity of mycoplasma infection the results were identical for the agar assay and PCR amplification. All positive cell lines displayed distinct, unequivocal, objectively discernible bands on agarose gels while the non-infected specimens showed no DNA amplification. A simultaneously performed comparison with four other commonly used detection methods (DNA-RNA hybridization in solution, DAPI DNA fluorescence staining, immunostaining with a monoclonal antibody and an ELISA) showed that PCR produced significantly less false-negative or false-positive results than all the other methods. Furthermore, in dilution experiments, PCR correctly detected the infecting mycoplasmas at the lowest level of 1/10(4) whereas the other assays were less sensitive. It is concluded that double-step PCR employing nested primers is superior to other mycoplasma detection methods in many respects: simplicity and speed, high specificity and extreme sensitivity, objectivity and accuracy.

Animals↗
PubMed1993

Dicistronic transcription units for gene expression in mammalian cells.

Dicistronic vectors utilizing the internal ribosomal entry site sequence of poliovirus as the intercistronic region were constructed for gene expression in mammalian cells. We have developed two monocistronic expression vectors which facilitate the creation of dicistronic expression plasmids. The dicistronic expression plasmids encode transcription units which allow the coordinated translation of the two genes. Using internal luciferase and secreted alkaline phosphatase, we show the correlated expression of both reporter genes and expression levels comparable to those achieved by the respective monocistronic expression vectors.

Alkaline Phosphatase↗
PubMed1993

Interferon regulatory factor 1 (IRF-1) mediates cell growth inhibition by transactivation of downstream target genes.

Interferon regulatory factor 1 (IRF-1) is a DNA-binding factor which recognizes regulatory elements in the promoters of interferon (IFN)-beta and some IFN-inducible genes. We observed that expression of transfected murine IRF-1 in different mammalian cell lines leads to down-regulation or stop of proliferation depending on the extent of expression. Expression of fusion proteins composed of IRF-1 and the hormone binding domain of the human estrogen receptor does not exhibit IRF-1 activity in the absence of estrogen. However, after estrogen treatment of the cells IFN-beta promoters are activated and the cells stop growing. As shown by expression of IRF-1 mutants both functions of the IRF-1-protein require DNA-binding and transcriptional activation. Since secreted factors including IFNs are not responsible for the anti-proliferative effect of IRF-1 we suggest that IRF-1 may be regarded as a negative regulator of cell growth which acts by activation of down-stream effector genes.

Animals↗
PubMed1993

The effect of positive and negative pH-gradients on the stability of small unilamellar vesicles of negatively charged phospholipids.

The stability of small unilamellar vesicles (SUV) made from negatively-charged phosphatidate by ultrasonication or pH-jump has been investigated. As criteria for the vesicle stability are used: (I) the bilayer integrity as judged from the permeability of the fluorescent probe carboxyfluorescein (CF) and (II) the susceptibility of the phospholipid vesicles to fusion as judged by gel filtration and freeze-fracture electron microscopy. Egg phosphatidate SUV (PA-SUV) whose internal cavity is in equilibrium with the dispersion medium are strictly speaking thermodynamically unstable by these criteria. They may, however, be regarded as stable from a practical point of view. CF-release is observed with a half-time of 14 days and also some vesicle fusion, particularly at low temperature (4 degrees C). The small effects observed, e.g., the small tendency of the vesicles to undergo fusion is probably due to the high surface charge density of PA bilayers. A main finding of this work is that the same positive pH-gradient which is used in the pH-jump method to drive the formation of SUV from large phosphatidic acid bilayer sheets has a stabilizing effect on the resulting PA-SUV. Stabilization is achieved by positive pH-gradients of about two pH-units or more with the pH of the external medium exceeding the pH of the vesicle cavity. Under these conditions, up to about 8 weeks no significant loss of entrapped CF and no fusion of SUV was observed both at 4 degrees C and room temperature. In contrast, a reverse or negative pH-gradient of several pH units applied to PA-SUV (with the external pH being lower than that of the vesicle cavity) destabilizes PA-SUV. Such a gradient can be shown to lead to a dramatic perturbation of the lipid bilayer packing as evident from a significant increase in CF permeability. The local perturbation of the phospholipid bilayer is accompanied by massive vesicle fusion which is prominent at low temperature (4 degrees C).

Drug Stability↗
PubMed1993

Computed tomography and fine-needle aspiration cytology for preoperative evaluation of cystic tumours of the pancreas.

Cystic neoplasms of the pancreas are rare. They may present as a mucinous (malignant or potentially malignant) tumour or as benign serous cystadenoma. Accurate preoperative diagnosis is therefore essential, as an asymptomatic serous tumour may safely be followed clinically for years, whereas mucinous tumours require aggressive surgery. Thirteen patients with cystadenoma and cystadenocarcinoma of the pancreas were reviewed and how best to improve the rate of preoperative diagnosis was considered. Fine-needle aspiration cytology and computed tomography enable accurate preoperative distinction between mucinous and serous cystic tumours of the pancreas.

Adult↗
PubMed1993

Interferon-beta promoters contain a DNA element that acts as a position-independent silencer on the NF-kappa B site.

The human interferon-beta (IFN-beta) promoter contains several functional domains that contribute to its virus-inducible regulation. One of them, PRDII, and NF-kappa B-binding sequence, can function as a constitutively activating element. Due to the presence of a negative regulatory domain that mediates a constitutive repression the natural IFN-beta promoter is silent in the non-induced state. Within this domain we have delimited an 11 bp element that acts as a negative regulatory element (NRE) of PRDII. Although the NRE is physically overlapping with PRDII in the IFN-beta promoter, it acts as a position-independent silencer of PRDII. Virus infection, which leads to the transcriptional activation of the IFN-beta promoter, does not alter the negative activity of the NRE on an isolated PRDII. It is the cooperative effect of PRDI and PRDII that is able to overcome the NRE function after virus infection. By UV cross-linking analysis using uninduced and virus-induced nuclear extracts, we show that two factors with molecular masses of approximately 95 and 100 kDa bind to the NRE.

Base Sequence↗