Analysis of larger than tetrameric poly(adenosine diphosphoribose) by a radioimmunoassay in nuclei separated in organic solvents.

Anitibodies were prepared against poly(adenosine diphosphoribose) of an average chain length of 40 adenosine diphosphoribose units by repeated injection of the polymer mixed with methylated albumin and adjuvants into rabbits. The antibody was present mainly in the 7 S fraction of the immunoglobulins. A membrane binding assay was developed, and its specificity determined for the detection of (adenosine diphosphoribose)ngreater than4 in organs. The method is suitable for the study of the variation of the polymer content of nuclei. The size recognition of the anti-poly(adenosine diphosphoribose) globulin fraction was the same for polymers composed of 4--40 adenosine diphosphoribose units, but smaller oligomers were not detectible. A quantitative extraction technique was developed and applied for radioimmunoassay of nuclear (adenosine diphosphoribose)n greater than 4. Organs were freeze-clamped, freeze dried, broken into subcellular fragments in a colloid mill, and the nuclear fraction was subsequently separated in organic solvents in order to preserve the polymer. Nicotinamide and nicotinic acid, when administered in vivo, augmented the (adenosine diphosphoribose)n greater than 4 content of rat liver and heart. Tissues of infant pigeons contained larger quantites of (adenosine diphosphoribose)ngreater than4 than tissues of adult rats.     

Molecular data on urinary glycoproteins with human leucocyte antigen (HLA) activity

Two glycoproteins characterized by their serological activities (HLA-A9 and HLA-B12), their isoelectric points and their molecular weights were purified from urine from a patient suffering from tubular proteinuria (cystinosis). Their physicochemical properties as well as an important increase of their specific activities during the different purification steps suggested that they behave as human leucocyte antigens (HLA) which had been excreted into urine. Their amino acid compositions and N-terminal sequences were different to those described for HLA solubilized from cultured human lymphoblast cell lines. The N-terminal sequences of the two serologically active glycoproteins were identical to the N-terminal sequence of another recently purified human urinary glycoprotein called human complex-forming glycoprotein. The relationship between HLA, human complex-forming glycoprotein and the serologically active urinary glycoproteins is discussed.     

Cytochalasin B and the kinetics of inhibition of biological transport: a case of asymmetric binding to the glucose carrier

Cytochalasin B inhibits glucose transport in human erythrocytes by competing with glucose for the carrier on the inner surface of the cell membrane, but there is no cytochalasin site associated with the outware-facing form of the carrier. Such asymmetry may be demonstrated by zero trans exit and entry experiments, whereas Sen-Widdas exit experiments are not easily interpretable. The orientation of the transport system appears to be reversed in certain other cell types: chich embryo fibroblasts, Novikoff hepatoma cells and HeLa cells. Here the cytochalasin site is present in the external but not internal carrier form.     

Prediction of the conformation of the cow and sheep kappa-caseins

The secondary structures of cow and sheep kappa-caseins were established according to the predictive rules of Chou and Fasman. The diagrams derived from this treatment allowed us to study the chymosin sensitive bond (milk-clotting process), as well as the glycosylation and phosphorylation sites, found to be situated in beta-turns. Despite a high variability between the primary structures of the COOH-terminal part (caseinoglycopeptide) of cow, sheep, and also other caseins, the secondary structures of the biologically important sites were found to be conserved.     

A new method for determining sulfoxides in peptide molecules using x-ray photoelectron spectroscopy

The problem of the quantitative determination of sulfoxide groups in peptide molecules has been re-examined. The approaches currently available for the estimation of δ-sulfoxide amino acids are limited in number and characterized by serious shortcomings; in addition, the choice of methods for the estimation of γ-sulfoxide amino acids is even more restricted. A new, rapid, and nondestructive direct method for determining quantitatively all types of sulfoxides in peptide molecules by using x-ray photoelectron spectroscopy is described.     

Determination of ADP-ribose and poly(ADP-ribose) by a new radioimmunoassay

A specific and sensitive radioimmunoassay for ADP-ribose has been developed on the basis of the selective conversion of ADP-ribose to 5'-AMP by alkaline treatment. Antibodies highly specific against 5'-AMP allowed quantification of ADP-ribose converted to 5'-AMP in the range of 1-40 pmol, and in the presence of large quantities of nucleic acids or 3'-AMP. Poly(ADP-ribose) could also be determined when degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. Determination of the chain length of purified polymer was possible by a parallel determination of ADP-ribose residues after glycohydrolase treatment and of 5'-AMP from the non-reducing end obtained by phosphodiesterase catalyzed hydrolysis. The high specificities of the alkaline conversion of ADP-ribose to 5'-AMP and of the radioimmunoassay for 5'-AMP allowed quantification of protein-bound ADP-ribose residues in crude tissue extracts as verified by comparison with chromatographically purified samples.     

Sister subchromatid exchanged segments and chromosome structure

The relationship between the synthesis of acidic nuclear proteins, phosphoproteins, RNA and chromatin ultrastructural pattern was studied in regenerating rat hepatocytes after partial hepatectomy. α-Amanitin induced, as early as 30 min after injection, a reduction of RNA synthesis to about 50% of the control level; the degree of inhibition had remained the same at 2 h after poisoning. No change was detected either in acidic nuclear protein synthesis or in phosphorylation for the whole time examined. The DNA-containing structures, demonstrated by the Gautier staining procedure, were in a dispersed pattern either in untreated regenerating hepatocytes or 30 min after α-amanitin administration to rats; but they did appear in a condensed form 1 h and more especially 2 h after toxin injection. In untreated regenerating hepatocytes, the regressive EDTA staining method for RNP revealed a large quantity of perichromatin fibrils which remained unchanged 30 min after α-amanitin treatment and were diminished at 1 h and strongly reduced 2 h thereafter.Cycloheximide treatment promptly reduced the synthesis of nuclear acidic proteins while leaving unchanged the synthesis of RNA; the quantity of perichromatin fibrils and the loosened appearance of DNA-containing structures were the same as in the control rat nuclei.Our results showed that the ultrastructural pattern of chromatin was not directly related either to the synthesis of RNA or to acidic nuclear proteins or to the phosphorylation of phosphoproteins; on the contrary, a strict relationship with the quantity of perichromatin fibrils was demonstrated. The possible interaction of perichromatin fibrils with other chromatin components was discussed as a possible regulatory mechanism of chromatin pattern.     

Sesquiterpenes from Lactarius blennius

Six sesquiterpene lactones were isolated from Lactarius blennius. The structures of two new sesquiterpenes, blennin A and blennin B were determinated by spectroscopic methods and the structure of the seco-compound, blennin C, is revised. The two known furan sesquiterpenes and lactarorufin A were also identified.