Amyloidogenicity of beta A4 and beta A4-bearing amyloid protein precursor fragments by metal-catalyzed oxidation.

Previously we have shown that the COOH-terminal 100 residues (A4CT) of the amyloid protein precursor (APP), which carry the sequence of the amyloid beta A4 protein of Alzheimer's disease at N-terminal position, form highly insoluble aggregates if expressed in the rabbit reticulocyte lysate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Dyrks, T., Weidemann, A., Multhaup, G., Salbaum, J.M., Lemaire, H.-G., Kang, J., Müller-Hill, B., Masters, C. L., and Beyreuther, K. (1988) EMBO J. 7, 949-957). Here we report that aggregation of this COOH-terminal APP fragment A4CT and also of beta A4 itself depends on additional factors. In contrast to the reticulocyte expression system, expression of A4CT and beta A4 in the wheat germ expression system resulted in only monomeric forms. We have identified the factors which are capable of transforming both soluble A4CT and beta A4 into insoluble and aggregating molecules. Monomeric A4CT or beta A4 expressed in the wheat germ lysate could be transformed into aggregating molecules by the addition of metal-catalyzed oxidation systems. The addition of radical scavengers such as ascorbic acid, trolox, and amino acids prevented the aggregation process induced by the radical initiators. Thus, the aggregation of amyloidogenic APP fragments if analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis requires amino acid oxidation and protein cross-linking induced by radical generation systems.     

Progestins induce down-regulation of insulin-like growth factor-I (IGF-I) receptors in human breast cancer cells: potential autocrine role of IGF-II.

Insulin-like growth factor-I (IGF-I) receptors are present in breast cancer cells and may play a role in breast cancer cell growth. We have studied the effect of progestins on IGF-I receptors in T47D human breast cancer cells. T47D cells constitutively express high levels of progesterone receptors and are a model for studying the regulation of cellular functions by progestins. Treatment of T47D cells with either progesterone or the synthetic progestin promegestone (R5020) decreased IGF-I receptor content by approximately 50%, as measured by Scatchard analysis and receptor biosynthesis studies. In contrast to progestins, estradiol, dexamethasone, and dihydrotestosterone did not influence IGF-I receptor content. No effect of R5020 was seen after 12 h of incubation, a near-maximal effect was seen after 24 h, and greatest effects were seen after 72 h. R5020 decreased IGF-I receptor mRNA abundance, indicating that progestins acted at the level of gene expression. However, progestins also increased the secretion of IGF-II, a ligand for the IGF-I receptor. In contrast to IGF-II, T47D cells did not express IGF-I. The addition of exogenous IGF-II to T47D cells down-regulated both IGF-I receptor binding and IGF-I receptor mRNA abundance. This study indicates, therefore, that progestins regulate IGF-I receptors in breast cancer cells and suggests that this regulation occurs via an autocrine pathway involving enhanced IGF-II secretion.     

Effect of insulin pump therapy on blood pressure and the renin-angiotensin system of diabetic rats

Insulin therapy, administered by continuous subcutaneous infusion with osmotic pumps over a 28 day period at doses of 2.5 and 5.0 units/day, resulted in a statistically significant increase in body weight of diabetic rats. The concentration of blood glucose was reduced by 68% to 109 mg/dl blood sugar by the higher dose of insulin and only partial control of diabetes was achieved by the lower dose (185 mg/dl blood sugar, -39%). Blood pressure was normalized by both doses of insulin. Elevated serum angiotensin converting enzyme activity and plasma renin activity, expressed as generated angiotensin I, were unaffected by the lower dose of insulin, but were reduced by 26% and 40%, respectively at the higher dose. These data suggest that elevated serum ACE and plasma renin activity, commonly found in the streptozotocin-diabetic rat, may not be primarily responsible for hypertension in this model.     

Polyribosomes from Pear Fruit: II. CHANGES OCCURRING IN PULP TISSUES DURING RIPENING AND SENESCENCE

Detailed analyses of polysome profiles from lyophilized pulp tissues of pear fruits (Pyrus communis L. cv. Passe-Crassane) at different stages of ripening and senescence, and estimates of the amount of polysomal-associated mRNA, lead to the conclusions that during senescence (ripening), the ripening and the over-ripening processes can clearly be separated and respectively linked to the first and the second increase in the large mRNA species. Ethylene synthesis which occurs at the beginning of a normal ripening at 15°C after a cold storage or an ethephon treatment is related to an increase in mRNA and ribosomal material found only in pulp tissues. Finally, we suggest that in the pear fruit, the sequence of events which occurs during senescence (ripening) is initiated by two systems which regulate ethylene biogenesis, and that the first system is efficient only at low temperatures (from 0 to 4°C).     

Distribution of DNase I-sensitive sites in simian virus 40 nucleoprotein complexes from disrupted virus particles.

Nucleoprotein complexes (core particles) released from simian virus 40 (SV40) virions were compared with similar complexes (SV40 chromatin) extracted from nuclei of infected cells. Core particles were sensitive to cleavage by DNase I at about the same enzyme concentration required to cleave SV40 chromatin. DNase I preferentially cleaved SV40 chromatin adjacent to the viral origin of replication; however, cleavage of core particles occurred with much less selectivity. The difference between these nucleoproteins was not due to a structural alteration induced by the virion disruption procedure, since SV40 chromatin retained its pattern of DNase I-sensitive sites when subjected top this treatment. On the other hand, core particles did not acquire the nuclease-sensitive feature typical of SV40 chromatin when they were exposed to infected cell nuclei and the Triton X-100-EDTA extraction procedure. Hence, the nuclease-sensitive feature was lost or altered during the normal process of virion assembly and maturation.     

Comparative immunochemical study of ribosomal proteins from various mammalian cells by two-dimensional immunoelectrophoresis

Summary Proteins isolated from ribosomal subunits of various mammalian cells were analysed comparatively by two different methods: a two-dimensional polyacrylamide gel electrophoresis system and a recently described two-dimensional immunoelectrophoresis technique. For this purpose, antisera were raised in rabbits against the total mixture of ribosomal proteins from murine cells. These sera were characterized by ring-test, double immunodiffusion and two-dimensional immunoelectrophoresis. They were shown to contain antibodies to a large number of ribosomal proteins. Immunoelectrophoretic analysis of 60S and 40S subunit proteins from rabbit, lamb, canine and human cells using anti-murine sera revealed a striking conservation of their antigenic properties. These results corroborated those obtained by two-dimensional polyacrylamide gel electrophoresis.     

Post-transformational rearrangement of an in vitro reconstructed group-A streptococcal erythromycin resistance plasmid

Cleavage of the group-A streptococcal macrolide, lincosamide, and streptogramin B (MLS) resistance plasmid pSM19035 yields 2 fragments [13 and 4 megadaltons (MD)] with EcoRI, and 15 fragments with HindIII, 12 of which are 6 pairs of identical fragments derived from the inverted repeats that comprise about 80% of the pSM19035 genome. The large EcoRI fragment was isolated, ligated, and used to transform the Challis strain of Streptococcus sanguis to erythromycin resistance. Plasmids (pDB101, pDB102, and pDB103) isolated from three different transformants had lower molecular masses than the original large EcoRI fragment. HindIII digestion of these molecules and subsequent analysis of fragment radioactivity distributions indicated the loss of plasmid segments of various sizes. The deletions, all of which occurred in the palindrome, did not affect the level and the inducible nature of pSM19035-determined antibiotic resistance. Only pDB101 retained the unique EcoRI cleavage site. The results of this analysis allowed the construction of an EcoRI and HindIII cleavage-site map of pSM19035 and promise to simplify future studies of genetic functions specified by streptococcal MLS resistance plasmids.