Determinants of growth-promoting activity reside in the A-domain of insulin-like growth factor I

A two-chain, disulfide linked, insulin-like compound embodying the A-domain of insulin-like growth factor I (IGF-I) and the B-chain of insulin has been synthesized and characterized with respect to insulin-like biological activity and growth-promoting potency. The compound displays a potency of ca. 41% relative to insulin in assays for insulin-like activity (e.g., lipogenesis) but significantly higher activity than insulin, ca. 730% relative to insulin, in growth factor assays (e.g., thymidine incorporation). The compound is, however, a less potent growth factor than IGF-I itself, ca. 26.5% relative to IGF-I, and is not recognized by IGF carrier proteins. We conclude that structural features contained in the A-domain of IGF-I are primarily responsible for the growth-promoting ability displayed by IGF-I, while features in the B-domain are responsible for recognition by IGF carrier proteins.     

The fate of cells with chromosome aberrations after total-body irradiation and bone marrow transplantation

Cytogenetic studies were done on bone marrow cells and peripheral lymphocytes of four patients (three with acute nonlymphocytic leukemia, one with aplastic anemia) at various intervals up to 861 days after total-body X irradiation (TBI) at doses between 4.5 and 10 Gy (450-1000 rad) followed by syngeneic or allogeneic bone marrow transplantation. Whereas no radiation-induced aberrations could be found in the bone marrow, apart from a transient finding in the patient with the lowest radiation dose, aberrant metaphases were seen in the peripheral lymphocytes of three patients in the range from 2.5 to 46% even at 861 days after the exposure. There were no demonstrable aberrations related to TBI in the only patient developing graft-versus-host disease. The dicentric yield as determined in the aberrant metaphases with 46 centromeres ranged between 3.4 +/- 1.3 and 4.9 +/- 0.4. In one patient it was demonstrated by BUdR-labeling that after 10 Gy (1000 rad) TBI the surviving and heavily damaged lymphocytes can go into cell cycle and reach at least the third mitosis. The percentage of aberrant cells diminished by about 25% at each mitotic division.     

Isolation of cDNA for bovine stomach 155 kDa protein exhibiting myosin light chain kinase activity.

Two proteins with myosin light chain kinase activity and electrophoretic molecular weights of 155,000 and 130,000 were each isolated from bovine stomach smooth muscle [Kuwayama, H., Suzuki, M., Koga, R., & Ebashi, S. (1988) J. Biochem. 104, 862-866]. The 155 kDa component showed a much higher superprecipitation-inducing activity than the 130 kDa component, when compared on the basis of equivalent myosin light chain kinase activity. In this study, we isolated a cDNA for the entire coding region of the 155 kDa protein. The deduced amino acid sequence revealed a high degree of similarity to those of chicken and rabbit smooth muscle myosin light chain kinases. Multiple motifs, such as three repeats of an immunoglobulin C2-like domain, a fibronectin type III domain, and unusual 20 repeats of 12 amino acids were detected in the sequence. Part of the amino-terminal sequence was similar to that of the actin- and calmodulin-binding domain of smooth muscle caldesmon. These observations suggest that the 155 kDa protein has additional functions other than its enzymatic activity. Two mRNAs of 6.0 and 2.6 kb in length in the bovine stomach smooth muscle RNAs were hybridized with cDNA probes. The 2.6-kb RNA probably encodes telokin, which is the carboxyl terminus of smooth muscle myosin light chain kinase. mRNAs with identical lengths were also detected in bovine aorta.     

Site-directed mutagenesis in the uracil-repair system. Preparation of mutated forms of human alpha 2 interferon

The mutant forms of human IFN-alpha 2 gene are obtained by oligonucleotide-directed mutagenesis with the use of uracil-repair system. To intensify the process the procedure of the uracil-containing DNA template preparation is modified. It was determined that when mutagenesis is performed in the uracil-repair system the yield of the process depends on the mutant DNA-strand in vitro synthesis efficiency. It is shown that the stability of the 5'-end primer-template complex and the level of the endogenic primers elongation are the basis factors, that determine induction mutations.     

Purification, characterization and immunolocalization of fimbrial protein from Porphyromonas (bacteroides) gingivalis.

Rapid and reproducible method is described here for the purification of the 43 kDa fimbrial protein from P. gingivalis by preferential fractionation in the presence of 1% SDS and 0.2M of a bivalent cation at pH 6.5. Homogeneity of the purified 43 kDa was confirmed by SDS-PAGE and Western blot analysis using monoclonal and polyclonal antibodies raised against this protein. Amino acid composition and the amino acid sequence of the first 30 amino acid residues of the purified fimbriae are consistent with the composition and sequence predicted from the cloned gene of the fimbrial subunit. Circular dichroism spectra shows high levels of beta-sheet structure. The purified 43 kDa polymer shows fimbriae-like morphology under the electron microscope. Ultrastructural localization of the 43 kDa protein by the immunogold technique revealed specific labeling of the fimbriae with a diameter of approximately 3.5 to 5.0 nm. Localization of this protein suggest that the 43 kDa component is a fimbrial subunit.     

Phosphorylating-dephosphorylating enzymes of avian oviductal secretions

Avian oviductal fluids contain phosphorylating-dephosphorylating enzymes that might function in sperm-oocyte interactions. Phosphorprotein phosphatase and protein kinase have been purified 20-fold and 40-fold, respectively. The latter easily aggregates and is highly labile. Other properties are comparable to those of holoenzymes.