Search PubMed⌕ Search

Biomedical subjects

K Weber

Publications and source records attributed to K Weber.

At least 703 records · Page 39Linked to original sources
PubMed1977

The isolation of tubulin and actin from mouse 3T3 cells transformed by simian virus 40 (SV3T3 cells), an established cell line growing in culture.

Tubulin can be purified from mouse SV3T3 cells (3T3 cells transformed by SV40 virus) by several cycles of temperature-dependent polymerization and depolymerization. Electron microscopical analysis of the final product reveals morphologically normal microtubules. Homogeneous actin can be isolated as a byproduct of the purification procedure. Mouse SV3T3 actin and skeletal muscle actin were compared by fingerprint analysis of the tryptic peptides obtained from performic-acid-oxidized protein. The two actins show a high degree of homology although apparently five of the twenty-five spots visualized by fluorescamine show a difference in chromatographic mobility. The purification procedure described allows the rapid isolation of both actin and tubulin from tissue culture in sufficient amounts for comparative biochemicals studies.

Actins↗
PubMed1977

Determination of oxytetracycline induced fluorescence and autofluorescence as an indicator of age and sex differences in rat femur.

Simple and rapid methods for the determination and detection of autofluorescence and oxytetracycline (OTC) induced fluorescence in powdered and intact bone (without previous chemical extraction of the antibiotic) were used to estimate age and sex related differences in the rat femur. The autofluorescence always had collagen characteristics; it increased with age and was not sex dependent. The intensity of OTC induced fluorescence decreased with age, being higher in males than in females. In the oldest animals, however a slight increase in the fluorescence intensity was observed, and sex differences disappeared. The spectrofluorometric measurements of intact femora indicate a higher OTC retention on the bone surface of young animals and adult males but not in adult females. The results indicate that the determination of OTC retention in the bone using our quantitative fluorometric method and the detection of OTC deposition at bone surface using a spectrofluorometric technique can be a sensitive and inexpensive tool for experimental studies of bone changes in physiological and pathological conditions.

Age Determination by Skeleton↗
PubMed1977

Degradation of nucleic acids in cell lysates by S1 nuclease in the presence of 9 M urea and sodium dodecylsulfate.

Single-strand-specific nuclease S1 from Aspergillus oryzae is shown to degrade DNA and RNA in lysates of HeLa cells in the presence of 9 M urea and sodium dodecylsulfate. Free dodecylsulfate inhibits S1 nuclease. However, if the detergent is complexed with proteins prior to the addition of the enzyme, S1 nuclease can degrade nucleic acids at dodecylsulfate concentrations which would inhibit the enzyme completely if no other proteins were present. In lysates prepared from HeLa cells by treatment with dodecylsulfate and urea, the detergent is complexed by cellular proteins and therefore S1 nuclease can be used to digest DNA and RNA. DNA can be completely degraded but, even after heat-denaturation, only 60% of the cellular RNA is converted into acid-soluble material. Analysis of the acid-insoluble RNA fragments by gel filtration reveals that the majority of the degradation products is approximately of tRNA size.

Aspergillus oryzae↗
PubMed1977

Isolation of amino-terminal fragment of lactose repressor necessary for DNA binding.

lac repressor can be dissected by trypsin into a homogenous tetrameric core (accounting for residues 60 to 347), carrying inducer binding activity, and the monomeric amino-terminal peptides ("headpieces") accounting for residues 1 to 59 and 1 to 51, respectively. This restriction of the action of trypsin on lac repressor is obtained in 1 M Tris-HCl (pH 7.5)-30% in glycerol at 25 degrees C since only the peptide bonds at lysine-59 and to a lesser extent after at arginine-51 are cleaved under these conditions. The headpieces can be purified by gel filtration. They have ordered secondary structure as revealed by circular dichroism studies. The monomeric headpieces show the relatively weak binding to nonoperator DNA but not the highly specific and strong binding to operator DNA typical for tetrameric lac repressor.

Amino Acid Sequence↗
PubMed1977

The display of microtubules in transformed cells.

Monospecific tubulin antibodies have been used in indirect immunofluorescence microscopy on a variety of well characterized, transformed cell lines grown in tissue culture. Networks of colcemid-sensitive fibers are seen in SV40-transformed 3T3 cells, SV40-transformed rat embryo cells, HeLa cells and other transformed cell lines. In each case, greater than 90% of the cells contain visible microtubular networks, and where individual microtubules can be distinguished, they run for long distances. Documentation of these metworks is more difficult in transformed than in normal cells, because transformed cells are in general more rounded and have less well spread cytoplasm. In addition, the microtubular networks can be readily visualized in "cytoskeletons" of both normal and transformed cells, obtained by treatment of cells with nonionic detergents in a buffer which stabilizes microtubules in vitro. Addition of calcium to this buffer results in in situ fragmentation and destruction of the microtubular network. In view of these results, we conclude that transformed cells contain significant numbers of microtubules, and that in transformed cells, as in normal cells, microtubules are arranged in networks.

Animals↗
PubMed1977

Reaction of the anastral mitotic apparatus of endosperm cells of the plant Leucojum aestivum with antibodies to tubulin from porcine brain as revealed by immunofluorescence microscopy.

Structures binding an antibody against tubulin from porcine brain were localized in the giant anastral mitotic apparatus of endosperm cells of the monocotyledonous plant, Leucojum aestivum, by indirect immunofluorescence microscopy. Both continuous and chromosomal spindle fibers were strongly stained. Postive fluorescence was also noted in polar cap regions and, in prometaphase stages, to some extent at the fragmented nuclear envelope. Intermingling and branching of subfiber elements was frequently noted.

Animals↗
PubMed1977

[The regulation of the circulatory system in the early postoperative period after open-heart surgery during physical therapy and orthostatic exercise (author's transl)].

17 patients, who underwent open heart procedures, were examined during and after standardized exercises at the first, second and third postoperative day. At 10 defined time intervals the following parameters were measured: arterial blood pressure, right and left atrial pressure and in the pulmonary artery, arterial and venous blood gases and cardiac output by thermodilution. Oxygen consumption and cardiac index were calculated. The results show that the majority of patients after open heart surgery are well able to adjust to additional work load even early postoperatively.

Adult↗
PubMed1977

Phalloidin-induced actin polymerization in the cytoplasm of cultured cells interferes with cell locomotion and growth.

Phalloidin, the toxic drug from the mushroom Amanita phalloides, was injected into the cytoplasm of tissue culture cells and the changes in intracellular actin distribution were followed by immunofluorescence microscopy with actin antibody. At low concentrations, phalloidin recruits the non- or less highly polymerized forms of cytoplasmic actin into stable "islands" of aggregated actin polymers and does not interfere with the preexisting thick bundles of microfilaments (stress fibers). Differential focusing shows that these islands of phalloidin-induced actin polymers occur at a level in the cytoplasm that is above the submembranous bundles of microfilaments present on the adhesive side of the cells. The pattern of cytoplasmic microtubules remains unaffected by the injection of phalloidin; however, filamin, a protein usually associated with actin in the cytoplasm, is also recruited into the islands. At higher phalloidin concentrations, contraction of the cell is observed. These results are discussed in the light of previous biochemical studies by Wieland and Faulstich and their coworkers [for a review see Wieland, T. (1977) Naturwissenschaften 64, 303-309] on the in vitro interaction of phalloidin with muscle actin, which have documented that phalloidin reacts stoichiometrically with actin, promotes actin polymerization, and stabilizes actin polymers. In addition, we show that microinjection of phalloidin interferes in a concentration-dependent manner with cell locomotion and cell growth. These results indicate that a well-balanced controlled reversible equilibrium between different polymerization states of actin may be a necessary requirement for cell locomotion and may also influence other cellular functions such as growth.

Actins↗
PubMed1977

In vitro assembly of pure tubulin into microtubules in the absence of microtubule-associated proteins and glycerol.

Microtubule protein from porcine cerebrum was fractionate into pure tubulin and microtubule-associated proteins by chromatography on phosphocellulose. In agreement with previous studies, pure tubulin does not form microtubules to a significant extent at 37 degrees in normal assembly buffers, which are characterized by a low concentration of Mg2+ ions. If, however, the Mg2+ concentration is raised to approximately 10 mM, rapid and extensive self-assembly of pure tubulin into microtubules is observed, provided the tubulin concentration is above 2.5 mg/ml. At a protein concentration of 3 mg/ml, the lag period is 1.5 min and the assembly process is virtually complete after 6 min at 37 degrees. These microtubules are like normal microtubules--sensitive to calcium ions, colchicine, and low temperature.

Animals↗
PubMed1977

Visualization of a system of filaments 7-10 nm thick in cultured cells of an epithelioid line (Pt K2) by immunofluorescence microscopy.

During our studies with antibodies against structural proteins of the cytoskeleton of eukaryotic cells we have observed that sera from many normal rabbits decorate a fiber system in cells of the established rat kangaroo cell line Pt K2. The display and organization of these fibers are different from those of microfilament bundles (decorated by antibody to actin) and microtubules (decorated by antibody to tubulin). This new fiber system can be further distinguished by its resistance to reorganization when cells are treated with Colcemid or cytochalasin B. The decoration of this fiber system is not detected if Pt K2 cells are fixed with formaldehyde. Such sera also appear to decorate swirls of perinuclear fibers in mouse Neuro 2a cells, and in mouse 3T3 cells treated with mitotic drugs. Comparison of the immunofluorescence pictures with electron microscopic data suggests that the sera are visualizing bundles of intermediate 7- to 10-nm filaments.

Cell Line↗
PubMed1977

Membranes of mammary gland. XII. Loosely associated proteins and compositional heterogeneity of bovine milk fat globule membrane.

Electrophoretic patterns of polypeptides of milk fat globule differed quantitatively depending on extent of washing during membrane preparation. This was due to selective loss of loosely associated, extrinsic membrane proteins. Major polypeptides with apparent molecular weights of 155,000 and 43,500 and membrane glycoproteins were released selectively during preparation of milk fat globule membranes. Evidence suggested that xanthine oxidase was a constituent of the selectively removed polypeptide fraction of apparent molecular weight 155,000. A major class of polypeptide with an apparent molecular weight of 62,500 was not extracted from milk fat globule membrane by treatment with dilute salts, ethylenediaminetetraacetic acid, or by nonionic and ionic detergent solutions. Milk fat globule membranes were separated into seven subfractions on isopycnic centrifugation in sucrose density gradients. Specific activities of the enzymes 5'-nucleotidase, xanthine oxidase, acid and alkaline phosphatases were similar or identical in all fractions. Electrophoretic analysis showed these seven subfractions had similar polypeptide profiles. Both phospholipid and total lipid content of subfractions were correlated inversely with fraction density. The results show that milk fat globule membrane is nearly homogenous in content of intrinsic membrane proteins and certain membrane-bound enzyme activities but is markedly heterogeneous with respect to buoyant density and lipid content.

Animals↗