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Biomedical subjects

M Müller

Publications and source records attributed to M Müller.

At least 721 records · Page 40Linked to original sources
PubMed1996

Intracellular, genetic or congenital immunisation--transgenic approaches to increase disease resistance of farm animals.

Novel approaches to modify disease resistance or susceptibility in livestock are justified not only by economical reasons and with respect to animal welfare but also by recent advancements in molecular genetics. The control or elimination of infectious pathogens in farm animals is historically achieved by the use of vaccines and drugs and by quarantine safeguards and eradication. Currently, research on the improvement of disease resistance based on nucleic acid technology focuses on two main issues: additive gene transfer and the development of nucleic acid vaccines. The strategies aim at the stable or transient expression of components known to influence non-specific or specific host defence mechanisms against infectious pathogens. Thus, candidates for gene transfer experiments include all genes inducing or conferring innate and acquired immunity as well as specific disease resistance genes. Referring to the site and mode of action and the source of the effective agent the strategies are termed 'intracellular', 'genetic' and 'congenital' immunisation. The targeted disruption (deletive gene transfer) of disease susceptibility genes awaits the establishment of totipotential embryonic cell lineages in farm animals. The cytokine network regulates cellular viability, growth and differentiation in physiological and pathophysiological states. The identification of the JAK-STAT pathway used by many cytokines for their intracellular signal propagation has provided not only new target molecules for modulating the immune response but will also permit the further elucidation of host-pathogen interactions and resistance mechanisms.

Animal Diseases↗
PubMed1996

Altered drug-stimulated ATPase activity in mutants of the human multidrug resistance protein.

The characteristics of P-glycoprotein (MDR1), an ATP-dependent drug extrusion pump responsible for the multidrug resistance of human cancer, were investigated in an in vitro expression system. The wild-type and several mutants of the human MDR1 cDNA were engineered into recombinant baculoviruses and the mutant proteins were expressed in Sf9 insect cells. In isolated cell membrane preparations of the virus-infected cells the MDR1-dependent drug-stimulated ATPase activity, and 8-azido-ATP binding to the MDR1 protein were studied. We found that when lysines 433 and/or 1076 were replaced by methionines in the ATP-binding domains, all these mutations abolished drug-stimulated ATPase activity independent of the MgATP concentrations applied. Photoaffinity labeling with 8-azido-ATP showed that the double lysine mutant had a decreased ATP-binding affinity. In the MDR1 mutant containing a Gly185 to Val replacement we found no significant alteration in the maximum activity of the MDR1-ATPase or in its activation by verapamil and vinblastine, and this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either. However, the Gly185 to Val mutation significantly increased the stimulation of the MDR1-ATPase by colchicine and etoposide, while slightly decreasing its stimulation by vincristine. These shifts closely correspond to the effects of this mutation on the drug-resistance profile, as observed in tumor cells. These data indicate that the Sf9-baculovirus expression system for MDR1 provides an efficient tool for examining structure-function relationships and molecular characteristics of this clinically important enzyme.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

On the mechanism of hyperacidification in lemon. Comparison of the vacuolar H(+)-ATPase activities of fruits and epicotyls.

Lemon fruit vacuoles acidify their lumens to pH 2.5, 3 pH units lower than typical plant vacuoles. To study the mechanism of hyperacidification, the kinetics of ATP-driven proton pumping by tonoplast vesicles from lemon fruits and epicotyls were compared. Fruit vacuolar membranes. H+ pumping by epicotyl membranes was chloride-dependent, stimulated by sulfate, and inhibited by the classical vacuolar ATPase (V-ATPase) inhibitors nitrate, bafilomycin, N-ethylmaleimide, and N,N'-dicyclohexylcarbodiimide. In addition, the epicotyl H+ pumping activity was inactivated by oxidation was reversed by dithiothreitol. Cold inactivation of the epicotyl V-ATPase by nitrate ( > or = 100 mM) was correlated with the release of V1 complexes from the membrane. In contrast, H+ pumping by the fruit tonoplast-enriched membranes was chloride-independent, largely insensitive to the V-ATPase inhibitors, and resistant to oxidation. Unlike the epicotyl inhibitors, and resistant to oxidation. Unlike the epicotyl H(+)-ATPase, the fruit H(+)-ATPase activity was partially inhibited by 200 microM vanadate. Cold inactivation treatment failed to inhibit H+ pumping activity of the fruit membranes, even though immunoblasts showed that V1 complexes were released from the membrane. However, cold inactivation doubled the percent inhibition by 200 microM vanadate from 30% to 60%. These results suggest the presence of two H(+)-ATPases in the fruit preparation: a V-ATPase and an unidentified vanadate-sensitive H(+)-ATPase. Attempts to separate the two activities in their native membranes on linear sucrose density density gradients were unsuccessful. However, following detergent-solubilization and centrifugation on a glycerol density gradient, the two ATPase activities were resolved: a nitrate-sensitive V-type ATPase that is also partially inhibited by 200 microM vanadate, and an apparently novel vanadate-sensitive ATPase that is also partially inhibited by nitrate.

Adenosine Diphosphate↗
PubMed1996

Purification and characterization of the acetate forming enzyme, acetyl-CoA synthetase (ADP-forming) from the amitochondriate protist, Giardia lamblia.

Giardia lamblia, an amitochondriate eukaryote, contains acetyl-CoA synthetase (ADP-forming), an enzyme known only from one other eukaryote (Entamoeba histolytica) and a few anaerobic prokaryotes. The enzyme has been purified about 350-fold. The activity in the direction of acetate formation was dependent on ADP and inorganic phosphate. The reverse reaction could not be detected. Succinyl-CoA, propionyl-CoA and dADP were utilized with lower efficiency. The enzyme did not utilize AMP plus PPi thus differs from the broadly distributed acetyl-CoA synthetase (AMP-forming). The enzyme is responsible for acetate production accompanied by ATP generation, thus plays an important role in G. lamblia metabolism.

Acetate-CoA Ligase↗
PubMed1996

Primary structure of a cytosolic malate dehydrogenase of the amitochondriate eukaryote, Trichomonas vaginalis.

The nucleotide sequence of a gene coding for a 37 kDa subunit of a cytosolic malate dehydrogenase of Trichomonas vaginalis was established. The sequence of a gDNA clone and a cDNA clone, which lacked seven amino-terminal codons, were identical, indicating an absence of introns from the gene. Cell fractionation combined with sequencing of peptide fragments of the purified enzyme showed that the gene codes for an expressed cytosolic enzyme. The derived amino acid sequence was closely related to cytosolic malate dehydrogenases from animals and plants and from the eubacteria Thermus aquaticus and Mycobacterium leprae and was more distant from the enzyme of mitochondria and from Escherichia coli and certain other eubacteria. In phylogenetic reconstructions this enzyme shared a most recent common ancestor with other cytosolic enzymes.

Amino Acid Sequence↗
PubMed1996

Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative.

Oncogenic activation of Abl proteins due to structural modifications can occur as a result of viral transduction or chromosomal translocation. The tyrosine protein kinase activity of oncogenic Abl proteins is known to be essential for their transforming activity. Therefore, we have attempted to identify selective inhibitors of the Abl tyrosine protein kinase. Herein we describe an inhibitor (CGP 57148) of the Abl and platelet-derived growth factor (PDGF) receptor protein-tyrosine kinases from the 2-phenylaminopyrimidine class, which is highly active in vitro and in vivo. Submicromolar concentrations of the compound inhibited both v-Abl and PDGF receptor autophosphorylation and PDGF-induced c-fos mRNA expression selectively in intact cells. In contrast, ligand-induced growth factor receptor autophosphorylation in response to epidermal growth factor (EGF), insulin-like growth factor-I, and insulin showed no or weak inhibition by high concentrations of CGP 57148. c-fos mRNA expression induced by EGF, fibroblast growth factor, or phorbol ester was also insensitive to inhibition by CGP 57148. In antiproliferative assays, the compound was more than 30-100-fold more potent in inhibiting growth of v-abl-transformed PB-3c cells and v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/MK cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. Furthermore, anchorage-independent growth of v-abl- and v-sis-transformed BALB/c 3T3 cells was inhibited potently by CGP 57148. When tested in vivo, CGP 57148 showed antitumor activity at tolerated doses against tumorigenic v-abl- and v-sis-transformed BALB/c 3T3 cells. In contrast, CGP 57148 had no antitumor activity when tested using src-transformed BALB/c 3T3 cells. These findings suggest that CGP 57148 may have therapeutic potential for the treatment of diseases that involve abnormal cellular proliferation induced by Abl protein-tyrosine kinase deregulation or PDGF receptor activation.

3T3 Cells↗
PubMed1996

YAC transgenesis in farm animals: rescue of albinism in rabbits.

The generation of transgenic mice with mammalian genes cloned in yeast artificial chromosomes (YACs) has generated great interest in the field of gene transfer into livestock. Many of the problems associated with standard transgenesis-such as lack of crucial regulator elements and position effects related to the integration site, which lead to variation in expression levels irrespective of the dose of the transgene-have been practically overcome. The large size of YAC-derived gene constructs (in excess of 1 Mb) facilitates the presence and transfer of all elements required for the faithful regulation of a gene. With the experiments discussed in this report, we have addressed the possibility of applying the obvious advantages of YAC transgenesis to farm animals. We have generated transgenic rabbits carrying a 250 kb YAC covering the mouse tyrosinase gene by pronuclear microinjection, and thus rescued the albino phenotype of the transgenic individuals. To date, this is the first demonstration of a successful transfer of large genetic units into the germ line of farm animals. This development might improve the occurrence of transgene expression at physiological levels and specific sites in livestock. YAC transgenesis therefore will be applied in genetic engineering, for example, in the production of pharmacologically interesting proteins encoded by large gene units and generating transgenic donors for xenotransplantation.

Albinism↗
PubMed1996

Phenylamino-pyrimidine (PAP) derivatives: a new class of potent and selective inhibitors of protein kinase C (PKC).

Phenylamino-pyrimidines represent a novel class of inhibitors of the protein kinase C with a high degree of selectivity versus other serine/threonine and tyrosine kinases. Steady state kinetic analysis of N-(3-[1-imidazolyl]-phenyl-4-(3-pyridyl)-2-pyrimidinamine (5), which showed potent inhibitory activity, revealed competitive kinetics relative to ATP. The adjacent H-bond acceptor of the pyrimidine moiety next to an H-bond donor of the phenylamine was found to be crucial for inhibitory activity. N-(3-Nitro-phenyl)-4-(3-pyridyl)-2-pyrimidinamine (7) preferentially inhibited PKC-alpha (IC50 = 0.79 microM) and not the other subtypes tested. The inhibition constants of PKC-alpha and the antiproliferative effect on T24 human bladder carcinoma cells showed a qualitative correlation, although with some exceptions.

Aniline Compounds↗
PubMed1996

Examination of the mononuclear phagocyte system in lupus-prone male BXSB mice.

During the course of lupus-like autoimmune disease male BXSB mice develop an increasing monocytosis in the peripheral blood. As we demonstrated previously, this monocytosis is parallelled by an expansion of a strain-specific high number of macrophage precursor cells (CFU-M) in the bone marrow of male mice. To test the hypothesis that the expanded mononuclear phagocyte system (MPS) may promote autoimmune disease in these mice the organ-associated macrophage system was examined. Our latest data show that an unusual expansion of CFU-M also appears in spleen and liver of male mice 2 weeks after birth. In addition to a morphological alteration of the organs during the course of the disease there is a change in number and distribution of organ-resident macrophages. Considering these results the possible contribution of the expanded MPS in promoting autoimmune disease is discussed.

Animals↗
PubMed1996

Acoustic distortion products from the cochlea of the blind African mole rat, Cryptomys spec.

The measurement of distortion-product otoacoustic emissions is a noninvasive method that can be used for assessing the sensitivity and the frequency tuning of nonlinear cochlear mechanics. During stimulation with two pure tones f1 and f2, the acoustic 2f1-f2 distortion was recorded in the ear canal of Cryptomys spec. to study specializations in cochlear mechanics that could be associated with the presence of a frequency expanded cochlear region between 0.8-1 kHz. In addition, a distortion threshold curve was obtained which describes relative threshold of nonlinear cochlear mechanics. Sensitive distortion thresholds could be measured for stimulus frequencies between 0.4 to 18 kHz with a broad minimum between 0.75 to 2.5 kHz. The distortion threshold curve extends to higher frequencies than previous neuronal data indicated. As a measure of mechanical tuning sharpness in the cochlea, suppression tuning curves of 2f1-f2 were recorded. The tuning curves reflected the typical mammalian pattern with shallow low frequency and steep high frequency slopes. Their tuning sharpness was poor with Q10dB values between 0.3 and 1.88. In the range of the frequency expanded region, the Q10dB values were below 0.5. This finding emphasizes that the presence of frequency expansion does not necessarily lead to enhanced mechanical tuning in the cochlea and one has to consider if in certain bat species with cochlear frequency expansion and particularly sharp cochlear tuning, the two phenomena may not be interlinked.

Acoustic Stimulation↗
PubMed1996

Immunolocalization of cathepsin D in pneumocytes of normal human lung and in pulmonary fibrosis.

Cathepsin D expression has been assessed by immunohistochemistry and immunoelectron microscopy in fetal, normal adult and injured lungs of human beings. In addition to the well known positivity of alveolar macrophages and the bronchial epithelial cells, normal type I and to a lesser extent type II pneumocytes showed a granular, cytoplasmic staining pattern. Using immunogold labelling of lowicryl embedded human lung, cathepsin D was present in lysosomes of epithelial cells. Double immunofluorescence labelling employing type I and type II specific antibodies or lectins confirmed the epithelial staining for cathepsin D. At the terminal sac period during lung development cathepsin D appears in the alveolar epithelium. In fibrotic specimens, enhanced immunoreactivity was found in epithelial and non-epithelial cells. Proliferative epithelial formations were strongly stained with cathepsin D antibodies, whereas detached, desquamated epithelial cells were weakly positive or negative. We suggest that cathepsin D plays a role in the remodelling process during fibrogenesis.

Adult↗
PubMed1996

Expression of CD44 isoforms during bleomycin-or radiation-induced pulmonary fibrosis in rats and mini-pigs.

The distribution of CD44s and CD44v molecules in normal and injured lung tissue of rats and mini-pigs was studied by examining the immunohistochemical binding of monoclonal antibodies against CD44 isoforms. We showed that the expression of CD44v and CD44s varies greatly among different pulmonary fibrosis samples and that some tissues express either enhanced expression of CD44s, particularly in the interstitium and on alveolar macrophages, or very low levels of CD44v in the alveolar epithelium. Normal type II pneumocytes expressed the CD44s and CD44v molecules at the basolateral aspect of the cell. Such localisation favours a role for CD44 in epithelial cell-fibroblast interaction during lung development and repair.

Animals↗
PubMed1996

Influence of different volume therapies on platelet function in the critically ill.

OBJECTIVE: Both albumin and synthetic colloids such as hydroxyethyl starch (HES) solution are used to optimize hemodynamics in the critically ill. The influence of different long-term infusion regimes on platelet function was studied. DESIGN: Prospective, randomized study. SETTING: Clinical investigation on a university hospital surgical intensive care unit. PATIENTS: Twenty-eight consecutive trauma patients (injury severity score > 15 points) and 28 consecutive nontraumatized surgical patients with sepsis. INTERVENTIONS: The patients received either 20% human albumin (HA trauma, n = 14; HA sepsis, n = 14) or 10% low-molecular-weight HES solution HES 200/0.5 (HES trauma, n = 14; HES sepsis; n = 14) for 5 days to maintain central venous pressure and/or pulmonary capillary wedge pressure between 12 and 16 mmHg. MEASUREMENTS AND RESULTS: Platelet function was assessed by aggregometry (= turbidimetric technique) using adenosine diphosphate 2.0 mumol/l, collagen 4 microliters/ml, and epinephrine 25 mumol/l as inductors. Arterial blood was sampled on the day of admission or the day of diagnosis of sepsis (= baseline value) and over the next 5 days. Standard coagulation parameters (antithrombin III, fibrinogen, partial thromboplastin time) were also measured. Total use of HES by the 5th day totalled 4870 +/- 990 ml in the trauma and 3260 +/- 790 ml in the sepsis patients (HA trauma: 1850 +/- 380 ml; HA sepsis: 1790 +/- 400 ml). Maximum platelet aggregation decreased significantly during the first 2-3 days after baseline in all groups. At the end of the investigation period, platelet aggregation variables had recovered and reached (or even exceeded) baseline values. Within the entire investigation period, the course of platelet aggregation variables did not differ significantly between HA and HES-treated patients irrespective of whether they were trauma or sepsis patients. CONCLUSIONS: Alterations in hemostasis may occur for several reasons in the critically ill. Human albumin is the preferred first-line volume therapy in patients at risk for coagulation disorders. With respect to platelet function, volume replacement with (lower-priced) low-molecular-weight HES solutions can be recommended in this situation without any risk.

Adult↗
PubMed1996

The effects of pentoxifylline on circulating adhesion molecules in critically ill patients with acute renal failure treated by continuous veno-venous hemofiltration.

OBJECTIVE: Circulating adhesion molecules appear to be excellent markers of endothelial activation in critically ill patients. Pentoxifylline (PTX) may limit sequelae of inflammation and subsequent endothelial activation by various mechanisms. The influence of PTX on the plasma levels of soluble adhesion molecules in critically ill patients undergoing continuous veno-venous hemofiltration (CVVH) was studied. DESIGN: Prospective, randomized, blinded study. SETTING: Clinical investigation in the surgical intensive care unit of a university hospital. PATIENTS AND PARTICIPANTS: Fourteen consecutive patients suffering from acute renal failure (ARF) with postoperative complications who received continuous pentoxifylline (CVVH-PTX) i.v. were compared with 14 patients with ARF who did not receive PTX (CVVH control group). INTERVENTIONS: Pump-driven CVVH was carried out with a blood flow ranging from 120 to 150 ml/min. All patients received fentanyl and midazolam continuously and were on mechanical ventilation. PTX (300 mg) was given as a loading dose, followed by continuous infusion of 1.2 mg/kg per h for the next 5 days. MEASUREMENTS AND RESULTS: From arterial blood samples, plasma levels of soluble adhesion molecules (endothelial leukocyte adhesion molecules [sELAM-1], and intercellular adhesion molecule-1 [sICAM-1], vascular cell adhesion molecule-1 [sVCAM-1], and P-selectin granule membrane protein [sGMP-140] were measured using enzyme-linked immuno-sorbent assays (ELISA). Measurements were carried out before the start of CVVH to establish baseline values and continued during the next 5 days. MAIN RESULTS: Eleven of the CVVH-PTX patients and 8 of the CVVH control patients survived during the investigation period. In the CVVH-PTX patients 2.4 +/- 0.3 g/day of PTX was given. At baseline, plasma levels of sELAM-1, sICAM-1, and sVCAM-1 were markedly higher than normal in both groups. In the CVVH control patients, all measured soluble adhesion molecules increased further during the study period (sELAM-1 from 90 +/- 22 to 134 +/- 30 ng/ml; sICAM-1 from 958 +/- 173 to 1460 +/- 209 ng/ml; sVCAM-1 from 1100 +/- 188 to 1804 ng/ml; sGM-140 from 499 +/- 102 to 688 +/- 121 ng/ml) (p < 0.05), whereas in the PTX-treated CVVH patients, plasma levels of all soluble adhesion molecules remained almost unchanged. The PaO2/FIO2 increased in the PTX-treated patients (from 209 +/- 67 to 282 +/- 58 mmHg) and remained almost unchanged in the CVVH control patients. CONCLUSION: Leukocyte/endothelial interactions play an important role in the inflammatory process. Circulating adhesion molecules may serve as markers of the extent of inflammation. Continuous i.v. administration of PTX was successful in blunting the increase of soluble adhesion molecules in critically ill patients undergoing CVVH. Whether these effects result from improved circulation at the microcirculatory level or from (direct or indirect) beneficial effects on endothelial cells warrants further controlled studies.

Acute Kidney Injury↗
PubMed1996

Does long-term continuous administration of pentoxifylline affect platelet function in the critically ill patient?

OBJECTIVE: The methylxanthine derivative pentoxifylline (PTX) is one of those promising substances which are under current investigation to modify or limit inflammatory response. Anti-aggregation activity has also been described that may contribute to the beneficial effects of this substance. Long-term effects on platelet function have not been elucidated yet. DESIGN: Prospective, randomized study. SETTING: Clinical investigation on a surgical intensive care unit of a university hospital. PATIENTS: 26 trauma patients and 26 patients suffering from sepsis secondary to major operations were consecutively studied. INTERVENTIONS: The patients prospectively received either 1.5 mg/kg per h pentoxifylline continuously for 5 days (after a loading dose of 600 mg) (trauma-PTX, n = 13; sepsis-PTX, n = 13) or saline solution as placebo (trauma-control; n = 13; sepsis-control, n = 13). MEASUREMENTS: On the day of admission (trauma patients) or day of the diagnosis of sepsis and at 12:00 p.m. during the next 5 days, platelet aggregation induced by adenosine diphosphate (ADP 2.0 mumol/l), collagen (4 microliters/ml), and epinephrine (25 mumol/l) was determined by a turbidimetric method from arterial blood samples. Standard coagulation screen was also monitored. MAIN RESULTS: In untreated trauma and sepsis patients, maximum platelet aggregation induced by all three agonists decreased during the first few days after inclusion in the study [trauma: ADP - 17.1 +/- 8.0 rel% (% change from baseline); sepsis: ADP -26.1 +/- 5.6 rel%]. In due course, maximum platelet aggregation recovered, reaching the baseline value or even exceeding it (trauma patients). In the PTX-treated patients, platelet aggregation was significantly less impaired (sepsis group: ADP -4.4 +/- 3.3 rel%) or even increased beyond baseline values in the first few days of the study (trauma group: ADP 16.1 +/- 8.0 rel%). Fibrinogen plasma levels were lower in the non-treated control groups (p < 0.05) than in the PTX groups. CONCLUSIONS: Continuous infusion of PTX for 5 days did not impair platelet function in critically ill patients. In both trauma and sepsis patients, the usual deterioration in platelet function was even attenuated, which may be due to the effects of PTX on cytokine release (e.g., reduction in tumor necrosis factor and interleukin-1), improvement in microcirculation, or additional fibrinolytic effects.

Adult↗
PubMed1996

The sequence and organization of the core histone H3 and H4 genes in the early branching amitochondriate protist Trichomonas vaginalis.

Among the unicellular protists, several of which are parasitic, some of the most divergent eukaryotic species are found. The evolutionary distances between protists are so large that even slowly evolving proteins like histones are strongly divergent. In this study we isolated cDNA and genomic histone H3 and H4 clones from Trichomonas vaginalis. Two histone H3 and three histone H4 genes were detected on three genomic clones with one complete H3 and two complete H4 sequences. H3 and H4 genes were divergently transcribed with very short intergenic regions of only 194 bp, which contained T. vaginalis-specific as well as histone-specific putative promoter elements. Southern blot analysis showed that there may be several more histone gene pairs. The two complete histone H4 genes were different on the nucleotide level but encoded the same amino acid sequence. Comparison of the amino acid sequences of the T. vaginalis H3 and H4 histones with sequences from animals, fungi, and plants as well as other protists revealed a significant divergence not only from the sequences in multicellular organisms but especially from the sequences in other protists like Entamoeba histolytica, Trypanosoma cruzi, and Leishmania infantum.

Amino Acid Sequence↗