Marijke M. Kwant

Elucidation of the sequence of canine (pro)-calcitonin. A molecular biological and protein chemical approach.

From the canine thyroid gland a calcitonin (CT) immunoreactive peptide was purified by successive aqueous acid acetone extraction, gel filtration and HPLC. Gas-phase sequencing of the purified peptide showed that the first 25 amino acids had 65% sequence homology with the amino-terminus of the human CT prohormone. A canine cDNA library was then made from the thyroid gland. A plasmid was isolated containing a sequence that is homologous to part of exon 3, and the complete sequence of exon 4 of the human mRNA encoding preproCT. From this cDNA the amino acid sequence of canine CT is predicted. In comparison with well-known CT sequences of other species, the strongest homology exists with bovine, porcine and ovine CT.

IGF-I and retinoic acid regulate the distribution pattern of IGFBPs synthesized by the canine mammary tumor cell line CMT-U335.

Stromal-epithelial interactions modulate growth and development in normal and neoplastic mammary gland. The release of IGF binding proteins (IGFBPs) by the stromal compartment of the mammary gland may play a modulating role in the IGF-mediated proliferation of mammary epithelium. Therefore, the IGFBP-expression pattern of the canine mammary tumor cell line U335 (CMT-U335), which has a mesenchymal phenotype, was determined. In addition, the effects of IGFs and all trans retinoic acid (RA) on DNA synthesis, and IGFBP secretion and distribution were examined. The IGFBPs secreted by CMT-U335 were characterized as IGFBP-2, -4, -5, and -6. Moreover, CMT-U335 appeared to be a suitable mammary mesenchymal cell line for study of the regulatory factors of IGFBP expression and the mechanism(s) involved. IGFs and RA enhanced IGFBP concentrations in cell-conditioned medium with IGF-I and RA having an additive effect. The IGF-I-stimulated DNA synthesis, however, was inhibited by RA. The difference between IGF-I and RA was an enhanced IGFBP-5 binding to the extracellular matrix (ECM) by RA, whereas IGF-I reduced binding to the ECM. Because high doses of insulin had no significant effects on IGFBP concentrations in the medium, it is concluded that IGF-I-induced changes in IGFBP concentrations are not mediated by type-I IGF receptors and may be the consequence of IGFBP redistribution.

Predicted primary and antigenic structure of canine corticotropin releasing hormone

Although the dog has been recognized as a useful model for the study of the cerebrospinal and peripheral actions of corticotropin releasing hormone (CRH) the exact amino acid composition of canine CRH is still unknown. In the present study the structure of canine CRH was predicted from the partial sequence of the gene encoding canine CRH. The CRH gene was amplified from genomic DNA obtained from white blood cells by a polymerase chain reaction and subsequently sequenced using the dideoxy method. The likely structure of canine CRH is: SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-NH2, which is identical to the structure of human, rat and equine CRH. PEPSCAN analysis of 3 different CRH antisera predicted an antiserum raised against a conjugate of human CRH and CNBr -activated thyroglobulin to be the antiserum of choice for the measurement of CRH in the dog. Preliminary data confirmed the existence of the highest cross-reactivity of a canine hypothalamus extract, known to have a high content of CRH, with antisera directed against human, rat CRH. As a result of the present study immunological tools for CRH estimations are characterized. Also, a homologous DNA probe for in situ hybridizations has become available for further investigations.

Adenovirus DNA replication in vitro is stimulated by RNA from uninfected HeLa cells

Adenovirus DNA replication was studied in a partially reconstituted system consisting of purified viral proteins (DNA‐binding protein, precursor terminal protein and Ad DNA polymerase) and a nuclear extract from uninfected HeLa cells. Optimal DNA replication required the presence of a heat‐stable, ribonuclease‐sensitive fraction from the cytosol of uninfected cells. This fraction stimulated the initiation about 3‐fold and the replication of origin fragments 5–10‐fold. Sedimentation analysis indicated the presence of a fast‐sedimenting and a slow‐sedimenting component which complemented each other. At least part of the stimulation was caused by low‐molecular‐mass RNA.

Replication in Vitro of Adenovirus DNA

The replication of the human adenovirus (Ad) DNA in infected cells is a highly efficient process. Within 40 hrs after infection a total amount of viral DNA is synthesized which almost equals the entire chromosomal DNA content of the cell. The abundance of replication proteins during infection has enabled the development of a replication system that initiates Ad DNA synthesis in vitro (1). So far, this is the only available system that replicates duplex DNA in higher eukaryotes. Since this system utilizes both viral and cellular proteins it may prove useful for the study of cellular DNA replication as well.

Adenovirus DNA Replication: Mechanism and Replication Proteins

The human adenoviruses, especially type 5 (Ad5) and type 2 (Ad2), can be easily cultivated in HeLa or KB cells. They have been a favorite subject for DNA replication studies in the eukaryotic cell during the last decade. The mature adenovirus genome isolated from virions consists of a linear duplex DNA molecule of about 35,000 base pairs. The DNA contains an inverted terminal repeat of 103 base pairs of which the first 50 nucleotides are very rich in A+T1,2. The inverted repeat is rather well conserved in the various adenoviruses and in particular base pairs 9–22 are identical in all human serotypes studied3.