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Biomedical subjects

R Albrecht

Publications and source records attributed to R Albrecht.

At least 37 records · Page 2Linked to original sources
PubMed1997

TPA induces apoptosis in MPC-11 mouse plasmacytoma cells grown in serum-free medium.

Apoptotic-like events could be rapidly induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in cells of the mouse plasmacytoma cell line MPC-11 grown in serum-free medium. Indicators for apoptosis were morphological changes visualized by light and electron microscopy, such as chromatin condensation and the formation of cellular buds and fragments, as well as biochemical indices like the appearance of the so-called 'DNA ladder'. Additionally, in these cells which are usually devoid of significant amounts of cytoplasmic intermediate filament (cIF) proteins, synthesis and accumulation of the cIF protein vimentin was rapidly induced by TPA treatment and almost all cells became vimentin-positive. Later on, substantial amounts of vimentin and lamin B degradation products appeared, and an increasing fraction of cells displayed low or even undetectable quantities of intact vimentin. This subpopulation was characterized via microscopy to be in the late stages of apoptosis. We suggest that in MPC-11 cells undergoing apoptosis in response to TPA treatment vimentin as well as lamin B are degraded, leading to a rearrangement and eventual loss of their respective filament networks.

Animals↗
PubMed1996

Myristoylated and non-myristoylated forms of the pH sensor protein hisactophilin II: intracellular shuttling to plasma membrane and nucleus monitored in real time by a fusion with green fluorescent protein.

Hisactophilins are myristoylated proteins that are rich in histidine residues and known to exist in Dictyostelium cells in a plasma membrane-bound and a soluble cytoplasmic state. Intracellular translocation of these proteins in response to pH changes was monitored using hisactophilin fusions with green fluorescent protein (GFP) and confocal laser scanning microscopy. Both the normal and a mutated non-myristoylated fusion protein shuffled within the cells in a pH-dependent manner. After lowering the pH, these proteins translocated within minutes between the cytoplasm, the plasma membrane and the nucleus. The role of histidine clusters on the surface of hisactophilin molecules in binding of the proteins to the plasma membrane and in their transfer to the nucleus is discussed on the basis of a pH switch mechanism.

Actins↗
PubMed1996

Specific suppression of human tumor necrosis factor-alpha synthesis by antisense oligodeoxynucleotides.

Recent clinical studies using neutralizing antibodies point to a key role for tumor necrosis factor-alpha (TNF-alpha) in chronic inflammatory diseases. Antisense technique is a recent approach aiming at inhibition of single proteins. Previously, we described nonspecific induction of TNF by phosphorothioate oligonucleotides. In this study, we established an in vitro model that allows specific inhibition of TNF synthesis, bypassing TNF induction. Freshly isolated human monocytes were incubated with oligonucleotides and the cationic lipid lipofectin in different ratios. TNF synthesis was stimulated with lipopolysaccharide and quantified by a specific radioimmunoassay (RIA). Among all sequences tested, one of the antisense oligonucleotides complementary to the translation initiation region of TNF mRNA (5'-CAT GCT TTC AGT CAT-3') revealed highest efficacy. At 2 microM, the antisense oligonucleotide inhibited TNF synthesis by up to 79%. A concentration as low as 250 nM of the antisense oligonucleotide was effective. Scrambled controls and controls with different, defined degrees of mismatches confirmed a sequence-specific action. Examination with confocal fluorescence microscopy showed a marked difference comparing lipofectin-mediated vs. spontaneous uptake. This study defines criteria that from the prerequisite necessary for design and application of antisense oligonucleotides against TNF in vivo.

Fluorescent Dyes↗
PubMed1996

The role of the cortical cytoskeleton: F-actin crosslinking proteins protect against osmotic stress, ensure cell size, cell shape and motility, and contribute to phagocytosis and development.

We generated Dictyostelium double mutants lacking the two F-actin crosslinking proteins alpha-actinin and gelation factor by inactivating the corresponding genes via homologous recombination. Here we investigated the consequences of these deficiencies both at the single cell level and at the multicellular stage. We found that loss of both proteins severely affected growth of the mutant cells in shaking suspension, and led to a reduction of cell size from 12 microns in wild-type cells to 9 microns in mutant cells. Moreover the cells did not exhibit the typical polarized morphology of aggregating Dictyostelium cells but had a more rounded cell shape, and also exhibited an increased sensitivity towards osmotic shock and a reduced rate of phagocytosis. Development was heavily impaired and never resulted in the formation of fruiting bodies. Expression of developmentally regulated genes and the final developmental stages that were reached varied, however, with the substrata on which the cells were deposited. On phosphate buffered agar plates the cells were able to form tight aggregates and mounds and to express prespore and prestalk cell specific genes. Under these conditions the cells could perform chemotactic signalling and cell behavior was normal at the onset of multicellular development as revealed by time-lapse video microscopy. Double mutant cells were motile but speed was reduced by approximately 30% as compared to wild type. These changes were reversed by expressing the gelation factor in the mutant cells. We conclude that the actin assemblies that are formed and/or stabilized by both F-actin crosslinking proteins have a protective function during osmotic stress and are essential for proper cell shape and motility.

Actinin↗
PubMed1996

Modulation of ultraviolet light-induced oxidative stress in mice skin related to dietary vitamin A and selenium intake.

Weanling female SKH1 hairless mice were randomly divided into 4 groups. Each group was fed a particular regimen for 20 weeks: 1) normal basal diet with 5 IU vitamin A/g and 0.45 microgram selenium/g, 2) vitamin A deficient, 3) selenium deficient, and 4) vitamin A plus selenium deficient. Three hours before being sacrificed, half of the animals were subjected to UV A + B irradiation (3 J/cm2). Superoxide dismutases (SOD), catalase, Se glutathione peroxidase activities were determined in dorsal skin homogenates, as well as the concentrations of GSH and of thiobarbituric acid-reactive substances (TBARS), the latter being an index of lipid peroxidation. Ultraviolet light altered the antioxidant defense of mouse skin tissue: GSH level, catalase and Se glutathione peroxidase activities were lowered and SOD was unequally enhanced according to the nutritional status. Vitamin A and Se deficiencies did not perceptibly aggravate the UV-induced oxidative stress, although the former enhanced the decline of catalase expression induced by the irradiation. Probably through an adaptive mechanism, both dietary deficiencies increased the skin reserve of antioxidant GSH and thus appear to modulate the effects caused by reactive oxygen species.

Animals↗
PubMed1995

Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein Tag.

Coronin is a protein involved in cell locomotion and cytokinesis of Dictyostelium discoideum. Here we show that coronin is strongly enriched in phagocytic cups formed in response to particle attachment. A fusion of coronin with green fluorescent protein (GFP) accumulates in the cups within less than 1 min upon attachment of a particle and is gradually released from the phagosome within 1 min after engulfment is completed. Phagocytic cup formation competes with leading edge formation and can be interrupted at any stage. When the cup regresses, coronin dissociates from the site of accumulation. TRITC-labeled yeast cells have been used to assay phagocytosis quantitatively in wild-type and coronin-null cells. In the mutant, the rate of uptake is reduced to about one third, which shows that coronin contributes to the efficiency of phagocytosis to about the same extent as it improves the speed of cell locomotion.

Actinin↗
PubMed1995

Chemoattractant-controlled accumulation of coronin at the leading edge of Dictyostelium cells monitored using a green fluorescent protein-coronin fusion protein.

BACKGROUND: The highly motile cells of Dictyostelium discoideum rapidly remodel their actin filament system when they change their direction of locomotion either spontaneously or in response to chemoattractant. Coronin is a cytoplasmic actin-associated protein that accumulates at the coritcal sites of moving cells and contributes to the dynamics of the actin system. It is a member of the WD-repeat family of proteins and is known to interact with actin-myosin complexes. In coronin null mutants, cell locomotion is slowed down and cytokinesis is impaired. RESULTS: We have visualized the redistribution of coronin by fluorescence imaging of motile cells that have been transfected with an expression plasmid containing the coding sequence of coronin fused to the sequence encoding the green fluorescent protein (GFP). This coronin-GFP fusion protein (GFP). This coronin-GFP fusion protein transiently accumulates in the front regions of growth-phase cells, reflecting the changing positions of leading edges and the competition between them. During the aggregation stage, local accumulation of coronin-GFP is biased by chemotactic orientation of the cells in gradients of cAMP. The impairment of cell motility in coronin null mutants shows that coronin has an important function at the front region of the cells. The mutant cells are distinguished by the formation of extended particle-free zones at their front regions, from where pseudopods often break out as blebs. Cytochalasin A reduces the size of these zones, indicating that actin filaments prevent entry of the particles. CONCLUSIONS: These data demonstrate that coronin is reversibly recruited from the cytoplasm and is incorporated into the actin network of a nascent leading edge, where it participates in the reorganization of the cytoskeleton. Monitoring the dynamics of protein assembly using GFP fusion proteins and fluorescence microscopy promises to be a generally applicable method for studying the dynamics of cytoskeletal proteins in moving and dividing cells.

Animals↗
PubMed1995

Hemoglobin-based oxygen carrier preserves submaximal exercise capacity in humans.

OBJECTIVE: The objective of this study was to evaluate the exercise capacity of subjects given an autologous transfusion or a polymerized bovine hemoglobin solution to define the pharmacodynamics and pharmacokinetics of a new hemoglobin-based oxygen carrier (HBOC-201). METHODS: Six normal healthy male subjects (ages 25 to 45 years) participated in this randomized, single-blind, two-way crossover study, which took place at Upjohn Research Clinics in Kalamazoo, Mich. A radial artery catheter was inserted in each subject before serial cardiac output and pulmonary function tests and phlebotomy of 15% blood volume (750 ml plus another 250 ml for study laboratories yields 1000 ml, or about 150 gm human hemoglobin). This was followed by isovolemic hemodilution with Ringer's lactate plus an autologous blood transfusion (or HBOC-201) and 1 week later 45 gm bovine hemoglobin of HBOC-201 (or autologous transfusion). Bicycle exercise stress tests to anaerobic threshold (approximately 65% of predicted maximum aerobic capacity) were done before phlebotomy and at approximately 45 minutes after the autologous transfusion or HBOC-201 infusion. RESULTS: Subjects had similar exercise and diffusion capacity but lower lactate levels (for up to 24 hours) during HBOC-201 (which paralleled plasma HBOC-201 levels) than during autologous transfusion periods. Oxygen use (uptake) and carbon dioxide production at rest were greater during the HBOC-201 infusion than during the autologous transfusion period. The half-life of HBOC-201 was about 23 hours. CONCLUSIONS: Exercise capacity and diffusion capacity were similar after HBOC-201 and autologous transfusion. HBOC-201 resulted in greater oxygen (or uptake) and carbon dioxide production and lower lactate levels compared with autologous transfusion. Under the conditions of the study, the physiologic effects of 1 gm bovine hemoglobin of HBOC-201 were similar to 3 gm human hemoglobin from autologous transfusion.

Adult↗
PubMed1995

Lack of beta 1 integrin gene in embryonic stem cells affects morphology, adhesion, and migration but not integration into the inner cell mass of blastocysts.

A gene trap-type targeting vector was designed to inactivate the beta 1 integrin gene in embryonic stem (ES) cells. Using this vector more than 50% of the ES cell clones acquired a disruption in the beta 1 integrin gene and a single clone was mutated in both alleles. The homozygous mutant did not produce beta 1 integrin mRNA or protein, while alpha 3, alpha 5, and alpha 6 integrin subunits were transcribed but not detectable on the cell surface. Heterozygous mutants showed reduced beta 1 expression and surface localization of alpha/beta 1 heterodimers. The alpha V subunit expression was not impaired on any of the mutants. Homozygous ES cell mutants lacked adhesiveness for laminin and fibronectin but not for vitronectin and showed a reduced association with a fibroblast feeder layer. Furthermore, they did not migrate towards chemoattractants in fibroblast medium. None of these functions were impaired in heterozygous mutants. Scanning electron microscopy revealed that homozygous cells showed fewer cell-cell junctions and had many microvilli not usually found on wild type and heterozygous cells. This profound change in cell shape is not associated with gross alterations in the expression and distribution of cytoskeletal components. Unexpectedly, microinjection into blastocysts demonstrated full integration of homozygous and heterozygous mutants into the inner cell mass. This will allow studies of the consequences of beta 1 integrin deficiency in several in vivo situations.

Animals↗
PubMed1995

Motility and substratum adhesion of Dictyostelium wild-type and cytoskeletal mutant cells: a study by RICM/bright-field double-view image analysis.

To investigate the dynamics of cell-substratum adhesion during locomotion, a double-view optical technique and computer-assisted image analysis has been developed which combines reflection interference contrast microscopy (RICM) with bright-field imaging. The simultaneous recording of cell-substratum contact and cell body contour has been applied to aggregation-competent cells of Dictyostelium discoideum. These cells are distinguished from cells at earlier stages of development by small areas of contact to a substratum. Three questions have been addressed in analysing the locomotion of aggregation-competent cells. (1) What is the relationship between changes in the shape of cells and their contact to a substratum during a chemotactic response? (2) What is the relationship between protrusion and retraction of the cell body, and between local attachment and detachment? (3) Are there differences between wild-type and mutant cells that lack certain cytoskeletal proteins? During a chemotactic response the front region of the amoeba can bend towards the gradient of attractant without being supported by its contact with a surface, which excludes the necessity for gradients of adhesion for the response. The finding that in locomoting cells protrusion of the leading edge often precedes retraction establishes a pioneer role for the front region. The finding that gain of contact area precedes loss provides evidence for the coordination of interactions between the cell surface and a substratum. For comparison with wild-type, aggregation-competent triple mutant cells have been used that lack two F-actin crosslinking proteins, alpha-actinin and 120 kDa gelation factor, and an actin filament fragmenting protein, severin. Disturbances in the spatial and temporal control of cytoskeletal activities have been unravelled in the mutant by RICM and quantified by cross-correlation analysis of attachment and detachment vectors. In order to detect these disturbances, it was essential to analyse cell locomotion on the weakly adhesive surface of freshly cleaved mica.

Animals↗
PubMed1995

The in vivo role of annexin VII (synexin): characterization of an annexin VII-deficient Dictyostelium mutant indicates an involvement in Ca(2+)-regulated processes.

Dictyostelium discoideum cells harbor two annexin VII isoforms of 47 and 51 kDa which are present throughout development. In immunofluorescence and cell fractionation studies annexin VII was found in the cytoplasm and on the plasma membrane. In gene disruption mutants lacking both annexin VII isoforms growth, pinocytosis, phagocytosis, chemotaxis and motility were not significantly impaired under routine laboratory conditions, and the cells were able to complete the developmental cycle on bacterial plates. On non-nutrient agar plates development was delayed by three to four hours and a significant number of aggregates was no longer able to form fruiting bodies. Exocytosis as determined by measuring extracellular cAMP phosphodiesterase, alpha-fucosidase and alpha-mannosidase activity was unaltered, the total amounts of these enzymes were however lower in the mutant than in the wild type. The mutant cells were markedly impaired when they were exposed to low Ca2+ concentrations by adding EGTA to the nutrient medium. Under these conditions growth, motility and chemotaxis were severely affected. The Ca2+ concentrations were similar in mutant and wild-type cells both under normal and Ca2+ limiting conditions; however, the distribution was altered under low Ca2+ conditions in SYN-cells. The data suggest that annexin VII is not required for membrane fusion events but rather contributes to proper Ca2+ homeostasis in the cell.

Animals↗
PubMed1994

Excessive dietary selenium decreases the vitamin A storage and the enzymatic antioxidant defence in the liver of rats.

This study investigated the influence of selenium intake, over 8 weeks, on vitamin A level and on enzymatic antioxidant defence in the liver of young rats. Deficient animals were fed a well-balanced diet but without selenite addition; the Se content of this diet which originated from natural Se content of ingredients was 0.05 mg/kg. Controls were fed the same diet with 0.40 mg/kg added Se. The two other groups received high levels of Se, 2.05 or 4.05 mg/kg. Excessive Se intake decreased the concentrations of retinol and retinyl palmitate in the liver. The linear regression analysis indicated a significant (P < 0.001) dose-dependent vitamin A decline. As expected, Se deficit lowered glutathione peroxidase activity. The highest Se excess decreased the enzymatic antioxidation: Zn,Cu superoxide dismutase, catalase, glutathione peroxidase activities. Data showed that high dietary Se can sometimes enhance carcinogenesis and our results suggest that it is best to be cautious in administrating Se to humans with the aim of preventing diseases.

Animals↗
PubMed1994

[Characterization of 24-hour survival rate and duration of survival of hydroxyethyl starch cryopreserved erythrocytes after autologous transfusion in the dog].

BACKGROUND: Cryopreservation of erythrocytes using hydroxyethyl starch (HES) as cryoprotecting additive could result in a nearly unlimited storage stability of preserved red cells. In addition, it would allow its immediate use for transfusion. In order to assess the therapeutic efficacy of erythrocytes cryopreserved with HES, their 24-hour post-transfusion survival and long-term survival was evaluated. MATERIALS AND METHODS: The experiments were carried out with dog erythrocytes as an animal model for human erythrocytes. To each of 6 German shepherd dogs a 15-ml sample of erythrocyte suspension, labeled with 51Cr (25 microCi) after thawing, was autologously injected. Caused by hemolysis 29% of the formerly cryopreserved erythrocytes have not been labeled. To each of 6 control animals 15 ml of a suspension of freshly drawn and 51Cr-labeled erythrocytes was injected. The 51Cr radioactivity in later taken blood samples was a measure for the number of injected erythrocytes having remained in the circulation until the moment of blood withdrawal. The effect of cryopreservation was assessed by comparison of the test group with the control group. RESULTS: In both groups 30% of the applied cells left the circulation within 30 min. This was effected by pharmacological enlargement of the dogs' spleen and not by hemolysis of the erythrocytes. After the first 24 h all of the cryopreserved labeled erythrocytes had survived to the same amount (> 95%) as the labeled fresh red cells. Between 12 h and 20 days after injection, in both groups the 51Cr activity decreased exponentially by 4.8 and 4.5%/d. This difference was not significant. The area under the curve amounted to 1253 and 1257% d, respectively. CONCLUSIONS: There exists a subpopulation of red cells that is destroyed by freezing stress. As a result the freed stroma would be a serious transfusion risk. All erythrocytes having survived the cryopreservation procedure resemble the fresh erythrocytes with regard to the in-vivo survival; their therapeutic efficacy is not impaired. In the context of in-vitro results with human erythrocytes it can be expected that at the present developmental state of the cryopreservation procedure at least 93% of the human erythrocytes cryopreserved with HES have a normal 24-hour and long-term post-transfusion survival.

Animals↗
PubMed1993

Effects of protein deficiency on lipid peroxidation in the small intestine and liver of rats.

This study investigated the influence of protein restriction on lipid peroxide content (thiobarbituric acid-reactive substances) and the intracellular antioxidant defence system in the small intestine and the liver. Weanling male Sprague-Dawley rats were divided into two groups: a low-protein group fed ad lib. a diet containing 6% casein for 4 wk, and a control group fed a diet containing 22% casein but restricted to the same dietary intake as that of the low-protein group. Compared with pair-fed controls, the protein-deficient rats exhibited a decrease in glutathione content in the small intestine and the liver. Thiobarbituric acid-reactive substances were increased in the intestine only. Data on the enzymatic antioxidant defence system in the protein-deficient animals showed an increase in catalase activity in the intestine but a decrease of this activity in the liver. The activity of selenium-dependent glutathione peroxidase was decreased in the liver and remained unchanged in the intestine. Superoxide dismutase was not modified by protein deficiency in either tissue. In control rats, enzyme activities were 6-43 times higher in the liver than in the intestine. The deleterious effects of protein deficiency appeared more marked in the intestine.

Animals↗
PubMed1993

Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility.

Coronin is an actin-binding protein in Dictyostelium discoideum that is enriched at the leading edge of the cells and in projections of the cell surface called crowns. The polypeptide sequence of coronin is distinguished by its similarities to the beta-subunits of trimeric G proteins (E. L. de Hostos, B. Bradtke, F. Lottspeich, R. Guggenheim, and G. Gerisch, 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:4097-4104). To elucidate the in vivo function of coronin, null mutants have been generated by gene replacement. The mutant cells lacking coronin grow and migrate more slowly than wild-type cells. When these cor- cells grow in liquid medium they become multinucleate, indicating a role of coronin in cytokinesis. To explore this role, coronin has been localized in mitotic wild-type cells by immunofluorescence labeling. During separation of the daughter cells, coronin is strongly accumulated at their distal portions including the leading edges. This contrasts with the localization of myosin II in the cleavage furrow and suggests that coronin functions independently of the conventional myosin in facilitating cytokinesis.

Actins↗