Purification of a high molecular weight form of epidermal growth factor from urine of breast cancer patients.

A high molecular weight form of epidermal growth factor (EGF) was detected by means of an EGF radio-receptor assay and an anchorage-independent growth assay in the urine of breast cancer patients. Preliminary data indicate that the activity of this growth factor is associated with lymph node status and tumor size and that the activity becomes reduced after removal of the primary tumor. The EGF-related polypeptide was purified to homogeneity by a combination of Sephadex G-25 and Bio Gel P-30 chromatography followed by binding to, and elution from, EGF receptor rich A431 cells.

Retardation of growth and epithelial differentiation in suckling mice by anti-EGF antisera.

Anti-EGF serum administered to newborn mice led to an increasing rate of growth retardation and body weight gain during the following period up to 5 weeks after birth, but sometimes to an effect found reversible after 3-4 weeks. In addition, striking delay of differentiation of skin epidermis and appendages and of intestinal Paneth's cells was ascertained about 4 weeks after birth. The findings point to neutralization of EGF or abolition of its effects by anti-EGF serum and, thus, substantiate the suggestion that EGF plays a physiological role during the early postnatal growth and differentiation.

Production and characterization of four monoclonal antibodies specific for human interferon-alpha-1 and -alpha-2.

Four monoclonal antibodies against HuIFN-alpha-1 and -alpha-2 were produced by the lymphocyte hybridoma technique. The screening for monoclonal anti-IFN antibodies was performed by two different procedures permitting the detection of neutralizing and non-neutralizing antibodies. The first antibody screening in the supernatants of hybridoma clones was carried out by an immunodot ELISA using a novel chromogen, 2-bromo-1-naphthol, for the immunoperoxidase reaction.

Temperature dependent pH-instability of some alpha-L-arabinofuranosidases.

In this paper are described alpha-L-arabinofuranosidases, partially purified from some moulds, with very high lability of their activity to weakly alkaline or neutral pH-values. This pH-lability strongly depends on temperature. The form of the inactivation curves suggests involvement of charge changes in this irreversible process.

Infrared data indicate U.U mispairs in rat liver 5S RNA.

The infrared spectra of ribosomal 5S RNAs of Escherichia coli and rat liver were measured under conditions where the structure of 5S RNA is preserved. The comparison of the spectra shows significant spectral differences for the U residues which resemble in their characteristics the infrared spectra of polyuridylic acid in its base paired state. This strongly indicates the presence of U.

Glucose measurement in diluted blood.

The sensitivity, measuring range and lifetime of enzyme electrodes using glucose oxidase sandwiched between dialysis membranes or alternatively in a polyurethane layer directly on the surface of a platinum electrode are compared. The GOD modified electrode exhibits the highest sensitivity. However, the signal depends strongly on the stirring rate.

Describing protein structure: a general algorithm yielding complete helicoidal parameters and a unique overall axis.

We present a general and mathematically rigorous algorithm which allows the helicoidal structure of a protein to be calculated starting from the atomic coordinates of its peptide backbone. This algorithm yields a unique curved axis which quantifies the folding of the backbone and a full set of helicoidal parameters describing the location of each peptide unit. The parameters obtained form a complete and independent set and can therefore be used for analyzing, comparing, or reconstructing protein backbone geometry.

ESR studies of structure and kinetics of radicals from hydroxyurea. An antitumor drug directed against ribonucleotide reductase.

Hydroxyurea (HU) is a clinically applied antineoplastic drug, which quenches tyrosine radicals in the active site of ribonucleotide reductase (RR) and inhibits DNA synthesis in proliferating cells. Under oxidizing conditions (Cu2+ or H2O2) long-lived radicals from HU have been found by ESR. The structure of HU radicals was established to be: (formula; see text).

Stability and reactivation of tyrosine radicals from ribonucleotide reductase in tumor cells studied by ESR.

Naturally occurring tyrosine radicals from the M2 subunit of ribonucleotide reductase (RR) have been recorded by ESR in proliferating ordinary Ehrlich-ascites (EA) tumor cells of mice. Tyrosine radicals are stable in EA cells at room temperature for 2 h. Up to 500 mW no microwave saturation occurs.

Direct comparison of electric field-mediated and PEG-mediated cell fusion for the generation of antibody producing hybridomas.

As an alternative to polyethylene glycol (PEG), electric field pulses offer, in theory, fusion conditions whose parameters are better controllable. In 1985 (1) we reported on the successful generation of hybridoma clones by means of electrofusion performed in a batch-type manner similar to that usually employed with PEG, and applicable to any type of antigens. Here we summarize the results of a series of fusions performed since then in which both electric field and PEG induced fusion were directly compared.

A simple determination of the sideness of the NH2-terminus in the membrane bound cytochrome P-450 LM2.

A simple and fast procedure for determining the sideness of the NH2-terminus in membrane proteins using FITC as label has been developed and applied to microsomal cytochrome P-450 LM2. The main advantages of FITC, which was shown not to penetrate lipid membranes, as compared to other labels are that it is fluorescent and that it can be used for manual microsequencing of proteins. By use of FITC it was directly shown that the NH2-terminus of P-450 LM2 is localized at the cytoplasmic side of the endoplasmic reticulum membrane.

EGF binding is quantitatively related to growth in node-positive breast cancer.

Number of mitoses and EGF binding were determined in parallel in biopsies of 27 lymph-node positive and of 23 lymph-node negative breast cancer patients. For node-positive patients the parameters for cell growth and EGF binding were quantitatively correlated by the equation y = P3 + P1(1- exp(- P2x]. For node-negative cases neither the non-linear model nor the linear approximation described the data unambiguously.

Purification of the growth-related protein p25 of the Ehrlich ascites tumor and analysis of its isoforms.

1. Two of the three isoforms of the growth-related protein p25 of the Ehrlich ascites tumor have been purified to homogeneity by giant two-dimensional polyacrylamide gel electrophoresis. 2.

A new monoclonal antibody (A46-B/B10) highly specific for the blood group H type 2 epitope: generation, epitope analysis, serological and histological evaluation.

A monoclonal antibody recognizing the blood group H type 2 antigen has been obtained from a BALB/c mouse immunized with MCF-7 (human mammary carcinoma) cells. The specificity of this antibody (A46-B/B10, IgM, kappa) has been identified by haemagglutination tests, immunohistochemistry, binding inhibition studies, and absorption experiments performed with synthetic oligosaccharides. The antibody is virtually nonreactive with H type 1 antigen or with closely related type 2 structures (e.

Relationship between sedimentation behaviour of DNA-protein complexes and DNA single- and double-strand breaks after irradiation with gamma-rays, pulsed neutrons and 12C ions.

The sedimentation behaviour of DNA-protein complexes was studied following irradiation of Chinese hamster cells (V79-4) and human lymphocytes over a wide dose range of 137Cs gamma-rays, pulsed neutrons and accelerated 12C ions. We have shown that the decrease of relative sedimentation velocity of the complexes at low doses is related to the occurrence of single-strand breaks in DNA. Rejoining of the breaks during a repair period increases the sedimentation velocity.

The production and radioimmunoassay application of monoclonal antibodies to fluorescein isothiocyanate (FITC).

Monoclonal antibodies (MoAbs) were produced against the fluorescence marker fluorescein isothiocyanate (FITC). FITC was used as a hapten to label different proteins and the anti-FITC MoAbs were used to identify these labelled proteins in a solid-phase radioimmunoassay and in cellular radioimmuno-binding assays for the demonstration of antigens and antibodies.

Production and ELISA application of bispecific monoclonal antibodies against fluorescein isothiocyanate (FITC) and horseradish peroxidase (HRP).

Hybrid hybridomas producing bispecific monoclonal antibodies reacting with both horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC) were obtained by fusing two hybridoma lines and selecting the fused cells using a fluorescence activated cell sorter (FACS). FITC was used to label different monoclonal antibodies and the bispecific antibodies acted as a linking agent between FITC-labelled antibody and the marker enzyme HRP. This system was used in enzyme immunoassays for the detection of different antigens.

Phosphorylation, anti-HIV activity and cytotoxicity of 3'-fluorothymidine.

3'-fluorothymidine (FddThd) is well phosphorylated to the 5'-triphosphate in various relevant cell-lines. This results in fairly stable levels of this compound without accumulation of the 5'-monophosphate to the extent described for 3'-azidothymidine (AzT). The di- and triphosphate of FddThd seems unable to influence the ribonucleotide reductase in permeable cells.

Temperature-induced phase transitions in proteins and lipids. Volume and heat capacity effects.

The partial specific heat capacity and volume of globular proteins and dispersions of phosphatidylcholines in aqueous solutions have been determined over a broad temperature range using a precise scanning microcalorimeter and a vibrational densimeter. It is shown that the temperature-induced, gel-to-liquid crystalline phase transition in phosphatidylcholines proceeds without a noticeable change in heat capacity but with a significant increase in the specific volume, whereas heat denaturation in proteins takes place without a noticeable change in the volume but with a significant increase in heat capacity. This principal difference between temperature-induced conformational phase transitions in proteins and lipids demonstrates clearly that heat denaturation of proteins, in contrast to the gel-to-liquid crystalline phase transition in lipids, cannot be regarded as a process similar to melting.

Analysis of hamster lymphomas for the presence of hamster papovavirus DNA.

The hamster papovavirus (HaPV) is a polyomavirus isolated from skin epitheliomas arising spontaneously in young Syrian hamsters. It can induce lymphomas and leukaemias in newborn hamsters. Although no virus particles are detectable by electron microscopy, high amounts of monomeric and oligomeric forms of extrachromosomal HaPV DNA molecules are found in the lymphoma cells.

Antibody production against BLV-p24 in calves following application of cell extracts from tumorous lymph nodes of cattle with enzootic bovine leukosis.

In the cell extract from tumorous lymph nodes of bovine leukosis virus (BLV)-infected cattle (tumour cell extract) and from lymph nodes of BLV-free cattle (control cell extract) neither the gp51 nor the p24 antigens were detectable. The tumour cell extract contained receptors for the BLV antigens gp51 and p24. The immunogenicity of the cell extracts was tested in calves.

Active site model of cytochrome P-450 LM2.

Based on (i) a detailed analysis of the physicochemical properties of selected benzphetamine derived substrates and (ii) the identification of Tyr-380 as active site residue trans to thiolate theoretical studies (computer aided molecular design) revealed a model of the substrate binding site of cytochrome P-450 LM2. The results indicate that substrates with a butterfly-like bulky conformation exhibit the highest intrinsic activity. Those substrates which preferably exist in an extended conformation are sterically hindered to intensively interact with the binding site which is demonstrated by computer graphics.

Oxygen limitation induces indirectly the synthesis of cytochrome P-450 mRNA in alkane-growing Candida maltosa.

Candida maltosa cells grown on hexadecane under oxygen limitation have an up to 6-fold higher cytochrome P-450 content in comparison to cells cultivated at oxygen saturation. We show by mRNA quantification using an in vitro translation system and subsequent specific immunoprecipitation that the cytochrome P-450 induction occurs mainly on the transcriptional level. The signal for induction may be the enhanced intracellular hexadecane concentration owing to a reduced hydroxylation capacity of the cytochrome P-450 at oxygen limitation.

The thermodynamic essence of the reversible inactivation of Na+/K+-transporting ATPase by various digitalis derivatives is relaxation of enzyme conformational energy.

This paper reports on the kinetic and thermodynamic parameters describing the interaction of selected digitalis derivatives with hog and guinea-pig cardiac (Na+ + K+)-ATPase (Na+/K+-transporting ATPase EC 3.6.1.