Identification of alpha 2-macroglobulin as a carrier protein for IL-6

In this report we demonstrate that alpha 2-macroglobulin (alpha 2M) is a carrier protein for IL-6. IL-6 was found to bind plasma proteins and an immunoblot analysis revealed that the complex between IL-6 and plasma proteins contains alpha 2M. Furthermore, purified alpha 2M bound IL-6. alpha 2M did not inhibit IL-6 activity or its binding to homologous receptor. IL-6 bound to alpha 2M retained its biologic activity and became resistant to treatment with proteases, although free IL-6 was easily degraded. These findings indicate that alpha 2M plays an important role as a carrier protein for IL-6 in serum and makes IL-6 produced at the local inflammatory site available to lymphocytes, hepatocytes, and hematopoietic stem cells, resulting in the induction of the coordinate systemic host defense reactions, such as immune response, acute phase reaction, and hematopoiesis.     

Phasmids as effective and simple tools for construction and analysis of gene libraries

Phasmid lambda pMYF131, a hybrid of phage lambda vectors and plasmid pUC19, was constructed. The phasmid and its derivatives were shown to be efficient vectors for construction and analysis of gene libraries in Escherichia coli cells. The lambda pMYF131 DNA molecule contains all the genes and regions essential for phage lytic development. The plasmid cannot be packaged either in the monomeric or the oligomeric form due to its specific length. Elongation of the DNA molecule by ligation with fragments of foreign DNA can make it packageable and this is easily detected by plaque formation. Hence, the procedures used to construct genomic libraries can be simplified by selection of only recombinant DNA molecules just at the time and on the basis of their packaging in vitro. The output of recombinant clones per vector molecule was several times higher for vector lambda pMYF131, compared to phage vector lambda L47.1AB, and attained 3 x 10(6) clones per micrograms DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. lambda pMYF131 contains nine unique restriction sites which allow the cloning of DNA fragments with blunt ends and of fragments with various types of cohesive ends, obtained by digestion with 14 prototype restriction enzymes. The maximal size of the cloned DNA fragments is approx. 20 kb for lambda pMYF131. Phasmid vectors were used to construct libraries of bovine, pig and quail genomes, and genomic libraries of 17 species of bacteria. Application of suitable methods allowed the identification 13 individual genes within these libraries.     

Photoaffinity labeling with dihydropyridine derivatives of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain

The characteristics of photoaffinity labeling with the calcium agonist [3H]Bay K 8644 (Bay) and the calcium antagonists [3H]nitrendipine (Nit) and (+)PN200-110 (PN) of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain were investigated. In all these crude membranes, [3H](+)PN (20 nM) was mainly photoincorporated into one protein band with a molecular weight of 30,000 - 41,000 Da. It was also incorporated into some other bands of all these crude membranes. The photoincorporation of [3H](+)PN into these crude membranes was inhibited by the presence of 20 microM unlabeled (+)PN. The photoincorporation of [3H](+)PN into these crude membranes depended on its dose and on the time of UV irradiation. No incorporation of [3H](+)PN was observed in the absence of UV irradiation. The incorporation was not affected by the presence of 1 mM CaCl2 and/or 0.15 M NaCl, but was significantly decreased by 20 microM (+)PN and slightly decreased by 20 microM (-)PN, 20 microM Bay, 1 mM diltiazem, or 1 mM verapamil. Namely, enantiomers of PN caused various extents of stereoselective inhibition of photoaffinity labeling by [3H](+)PN of specific protein bands in these crude membranes. [3H]Nit was photoincorporated into these crude membranes in the same way as [3H](+)PN, but [3H]Bay was not photoincorporated. However, 20 microM unlabeled Nit did not consistently inhibit photoaffinity labeling with [3H]Nit. These findings suggested that measurement of photoaffinity of crude membranes from rat skeletal, cardiac, and uterine muscles and whole brain with [3H](+)PN by UV irradiation is a useful method for investigating the characteristics of the voltage-dependent calcium channels that are affected by 1,4-dihydropyridine derivatives.     

A monoclonal antibody that triggers deacylation of an intermediate thrombin-antithrombin III complex

Upon incubation of antithrombin III with thrombin in the presence of a monoclonal antibody recognizing an epitope exposed on the heavy chain part of thrombin-cleaved two-chain antithrombin III, antithrombin III was preferentially cleaved by the enzyme as a substrate, rather than covalently complexed with the enzyme to form an equimolar, stable acyl complex. Once the stable acyl complex was formed between the enzyme and antithrombin III, however, no further liberation of two-chain antithrombin III was observed. Kinetic studies showed that heparin does not affect this reaction, although generation of thrombin-cleaved two-chain antithrombin III is apparently accelerated in accordance with the rate constant for heparin-enhanced thrombin-antithrombin III complex formation. Here we propose the term "switching antibody" for an antibody that triggers deacylation of an intermediate enzyme-inhibitor complex by switching the enzyme-inhibitor reaction from the major pathway of stable acyl complex formation to an alternative pathway of cleavage of the inhibitor as a substrate.     

Adaptive response to 2 low doses of X-rays in human blood lymphocytes

Human peripheral blood lymphocytes exposed to a single adaptive dose of 1 cGy X-rays or 2 adaptive doses, each of 1 cGy, were found to be equally resistant to the induction of chromosome damage by subsequent challenge with a high dose of 1 Gy X-rays, as compared to cells that were not pre-exposed. They responded with a significantly reduced incidence of chromatid and isochromatid breaks. These results indicate the presence of an inducible chromosomal repair mechanism in human blood lymphocytes and confirm the observations made by earlier investigators. The incidence of chromosome damage was found to be similar in the lymphocytes pre-exposed to a single or 2 adaptive doses, suggesting that, under the conditions tested, the second adaptive dose did not offer any additional protection against the chromosome damage induced by the challenge dose.     

Proteolytic Activity against Model Peptides of the Cell Wall Lytic Enzyme from Chlamydomonas

A cell wall lytic enzyme (gamete wall-autolysin) from Chlamydomonasreinhardtii specifically cleaved several synthetic model peptides,-neo-endorphin, dynorphin (1–13), neurotensin and mastoparan,at the peptide bonds between consecutive hydrophobic amino-acidresidues. The cleavage was not significantly affected by high-saltconditions which are known to inhibit digestion of the cellwall. (Received December 14, 1989; Accepted April 5, 1990)     

Synthesis of aminosterols and their derivatives

Reactions of steroidal epoxides such as 5,6 alpha-epoxy-5 alpha-cholestane (I) and its 3 beta-chloro (II) and 3 beta-acetoxy (III) analogs with urea in dimethylformamide afforded 6 beta-amino-5 alpha-cholestan-5-ol (IV-VI), 6 beta-amino-N-formyl-5 alpha-cholestan-5-ol (VII-IX), and 6 beta-amino-N-amido-5 alpha-cholestan-5-ol (X-XII), along with the 5 alpha-cholestane-5,6 beta-diol (XIII-XV). In addition to these compounds, the 3 beta-acetoxy analog also afforded the N-carboxyl derivative (XVI).     

Distributions of antibovine-pepsinogen-positive cells in glandular regions of forestomach in the bactrian camel, Camelus bactrianus.

The present paper describes mucous cells in the forestomach glandular region which immunoreacted to antibovine pepsinogen serum in adult and young bactrian camels, Camelus bactrianus (female, 25 years old; female, 6 months old; from Hilar Inner Mongolia, People's Republic of China, in August 1988). These immunoreactive cells in forestomach glandular regions also reacted to Alcian blue (pH 1.0) and/or periodic acid-Schiff. Cross-reactivity of antibovine pepsinogen serum against extracts from the forestomach glandular region was analyzed by Western blotting, supporting that this serum could detect pepsin and/or pepsinogen in the bactrian camel. It was suggested that mucous cells in forestomach glandular regions produce pepsinogen molecules.