Patterns of expression of tumor markers on non-transformed human mammary epithelial cells cultured in vitro.

We have examined the expression of 7 well defined tumor markers/tumor associated antigens (H type 2, X, Y, sialyl-Lea, CEA, MAM-6, and Tn) and a tumor associated antigen defined by a new own monoclonal antibody on non-transformed human epithelial cell lines derived from reduction mammoplasties by means of immunocytochemistry with monoclonal antibodies. Two cell types are discernible: slowly or non-proliferating, lumenal derived (I), and proliferating, stem cell-like, basal cell-derived cells (II). Five out of the 8 tumor markers were expressed on type I cells, and all 8 on type II cells.

Structure of the hydration shells of oligo(dA-dT).oligo(dA-dT) and oligo(dA).oligo(dT) tracts in B-type conformation on the basis of Monte Carlo calculations.

Monte Carlo simulations [(N, V, T)-ensemble] were performed for the hydration shell of poly(dA-dT).poly(dA-dT) in canonical B form and for the hydration shell of poly(dA).poly(dT) in canonical B conformation and in a conformation with narrow minor groove, highly inclined bases, but with a nearly zero-inclined base pair plane (B' conformation).

Hydroxyl radical scavenging and antipsoriatic activity of benzoic acid derivatives.

The hydroxyl radical scavenging and antipsoriatic activity of a number of lipophilic and hydrophilic benzoic acid derivatives was investigated. To quantify antioxidative effects, a newly introduced test system based on the diminution of the ESR signal of DMPO-OH (generated by Fenton's reagent) by the tested compounds was applied. It was found that the in vitro antioxidative (toward hydroxyl radical) activity of benzoic acid esters decreases with increasing chain length whereas the antipsoriatic activity increases.

Quenching of tyrosine radicals of M2 subunit from ribonucleotide reductase in tumor cells by different antitumor agents: an EPR study.

The inhibition of ribonucleotide reductase (RR) of intact Ehrlich ascites tumor cells by different antitumor agents was compared using EPR spectroscopy. The inactivation of M2 subunit was measured via quenching of the functionally essential tyrosine radical. Inhibitors of different classes, for example, hydroxyurea, pyrogallol, and thiosemicarbazones, differ in their efficiency by three orders of magnitude.

Use of monoclonal antibody against major internal protein p24 of bovine leukemia virus in capture ELISA.

A monoclonal antibody (4H4) against the major internal protein, p24, of bovine leukemia virus (BLV) is described. It recognizes a sequence determinant on the p24-molecule and displays high affinity to its antigen. The monoclonal antibody 4H4 was applied in capture ELISA for diagnosis of enzootic bovine leukosis, using crude BLV-preparation as antigen.

Synthesis of bovine leukemia virus antigens in Escherichia coli.

Plasmids were constructed by the use of pEX vectors that encode and express different parts of the bovine leukemia virus (BLV): main core protein p24, nucleic acid-binding protein p12, transmembrane protein gp30, and different segments of envelope protein gp51. Expression of fusion proteins with molecular weights higher than 117 kD for all recombinant plasmids was shown in Coomassie-blue stained gels and by Western blot analysis with rabbit anti-BLV sera. Coupling of a gp51-encoding with a p24-encoding DNA fragment in pEX vectors led to synthesis of a fusion protein that was recognized by monoclonal antibodies directed against gp51 and p24 epitopes.

Recombinants from a proviral bovine leukemia virus genome corresponding to the 3' region transactivate viral LTR in NIH3T3 and non-infected FLK cells.

Bovine leukemia virus (BLV), like human T-cell leukemia viruses, Types I and II, contains three open reading frames at the 3' end of its genome. The longest open reading frame encodes a transactivator protein which is generated by a doubly-spliced mRNA. A series of co-transfection experiments, using proviral BLV pX expression plasmids under the control of the Moloney leukemia virus LTR and the indicator plasmid containing the assayable lac Z gene under the control of BLV LTR, revealed that both NIH3T3 cells and non-infected fetal lamb kidney cells are able to express an active transactivator protein.

Amperometric bi-enzyme based biosensor for the detection of lactose--characterization and application.

Based on the glucose oxidase-beta-galactosidase sequence an enzyme probe for the specific determination of lactose has been developed. beta-Galactosidases from different sources have been compared, the sensor containing beta-galactosidase from Curvularia inaequalis has been characterized in respect of optimal pH, enzyme loading, apparent activity and functional stability. The response of the bi-enzyme probe depends linearly on lactose concentration between 0.

Role of lipid in the electron transfer between NADPH-cytochrome P-450 reductase and cytochrome P-450 from mammalian liver cells.

1. The anaerobic NADPH-reduction of the isozymes cytochrome P-450 LM2 and LM4 was used as a functional tool to study the component interaction in reconstituted monooxygenase systems in dependence on different phospholipids. 2.

Demethylation of tertiary amines by a reconstituted cytochrome P-450 enzyme system: kinetics of oxygen consumption and hydrogen peroxide formation.

Initial reaction rates of oxygen consumption and hydrogen peroxide formation in a cytochrome P-450 catalyzed reaction are practically independent of the nature of tertiary amines that were used as substrates. From the kinetic studies and the substrate conversion results that the amount of water formed in a side reaction is determined by the substrate specificity. Both hydrogen peroxide and water formation lower the efficiency of the monooxygenatic activity of cytochrome P-450.

Generation of free radicals by photoexcitation of pheophorbide alpha, haematoporphyrin and protoporphyrin.

An investigation of the production of radical species by photoexcitation pheophorbide alpha, haematoporphyrin and protoporphyrin was performed. In an aqueous solution containing different amounts of ethanol, the superoxide radical was detected by the spin trapping technique. In addition, secondary radicals were observed.

The growth-related protein P23 of the Ehrlich ascites tumor: translational control, cloning and primary structure.

p23 is a protein of Ehrlich ascites tumor cells, preferentially synthesized in the exponentially growing tumor. In vitro, serum and actinomycin D rapidly induce p23 synthesis. Using transcription inhibitors and a wheat germ cell-free translation system, evidence is provided that the synthesis of p23 is under translational control.

Genetic analysis of DNA from single human oocytes: a model for preimplantation diagnosis of cystic fibrosis.

Gene sequences in human oocytes were studied to investigate the possibility of diagnosing inherited or sporadic genetic disease before implantation after in vitro fertilisation. By specific amplification the possibility of analysing the DNA from single human oocytes for a specific gene was shown, and genotypes for markers closely linked to cystic fibrosis and Duchenne muscular dystrophy were determined. Single oocytes were used to approximate the total amount of DNA present in a single cell taken for biopsy from a 4-16 cell blastocyst.

A novel L-glutamate oxidase from Streptomyces endus. Purification and properties.

A new flavoenzyme using molecular oxygen to oxidize L-glutamic acid has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from the culture medium of Streptomyces endus. Hydrogen peroxide, 2-oxoglutaric acid and ammonia are formed as products. Among 25 amino acids tested including D-glutamic acid, L-glutamine and L-aspartic acid, only L-glutamic acid is converted.

Molecular cloning and characterization of the primary structure of the alkane hydroxylating cytochrome P-450 from the yeast Candida maltosa.

A cDNA library was established starting from poly(A) RNA of n-alkane-grown Candida maltosa cells and cDNA clones were isolated containing the entire coding sequence for the alkane hydroxylating cytochrome P-450. The deduced protein consists of 521 amino acids, contains two putative transmembrane segments in the N-terminal region and has a characteristic heme-binding sequence in the C-terminal part. Sequence alignments with members of 11 reported cytochrome P-450 families revealed a strong homology to an alkane-inducible cytochrome P-450 from Candida tropicalis.

A new neomycin phosphotransferase II solid phase assay in combination with polyacrylamide sodium dodecylsulphate gel electrophoresis.

A new general method for the determination of neomycin phosphotransferase (NPT) II (EC 2.7.1.

Hydrodynamic and circular dichroic analysis of mammary-derived growth inhibitor (MDGI).

The mammary-derived growth inhibitor exists in solution as a monomeric molecule with a molar mass of 14,500 +/- 400 g/mol. The largest diameter and the height of the polypeptide chain were estimated to be 3.75 +/- 0.

EMBOPRO--an automatically generated protein sequence database.

For the identification of newly sequenced proteins it is necessary to have a large stock of known proteins for comparison. In this paper we present an automatically generated protein sequence database. The translation program introduced allows a periodical translation of every new release of the EMBL database.

Molecular cloning, sequencing and expression in Escherichia coli of the 25-kDa growth-related protein of Ehrlich ascites tumor and its homology to mammalian stress proteins.

The growth-related 25-kDa protein (p25) of Ehrlich ascites tumor (EAT) has been characterized by molecular cloning and sequencing of cDNA clones detected by hybridization with oligonucleotide probes synthesized according to the amino acid sequence of a tryptic peptide of p25. Detection of p25 mRNA in EAT of the exponential growth phase and of the stationary phase using cDNA-derived RNA probes demonstrated that the abundance of p25 mRNA is also growth-related. High-level expression of p25 in Escherichia coli has been established by oligonucleotide-directed mutagenesis of cDNA and insertion of the mutated cDNA into a T7-promoter expression vector.

A novel carbonochloridate for activation of supports containing hydroxyl groups.

The conditions for the introduction of active carbonate groups into supports containing hydroxyl groups by reaction with 5-norbornene-2.3-dicarboximido carbonochloridate are described. Up to 1.

Polarity as a criterion in protein design.

Hypothetical proteins can be tested computationally by determining whether or not the designed sequence-structure pair has the characteristics of a typical globular protein. We have developed such a test by deriving quantities with approximately constant value for all globular proteins, based on empirical analysis of the exposed and buried surfaces of 128 structurally known proteins. The characteristic quantities that best appear to segregate badly designed or deliberately misfolded proteins from their properly folded natural relatives are the polar fraction of side chains on the protein surface and, independently, in the protein interior.