Cloning and antigenic expression of two genes from Chlamydia psittaci avian strain

Abstract: Genes from Chlamydia psittaci P-1041 were cloned into the Bam HI site of pUC19 and were transformed to host Escherichia coli JM109. Two recombinant plasmids that expressed protein antigens of Chlamydia were isolated. The sizes of the DNA fragments were 1350 and 1710 bp, and encoded for polypeptides of M _r 25 and 42 kilodaltons (kDa), respectively. The 25-kDa protein had cross-reactivity with antisera to ten C. psittaci strains and two C. trachomatis strains, whereas the 42-kDa protein reacted only with homologous antiserum to the C. psittaci P-1041 strain. Furthermore, in Southern hybridization analysis these two fragments as probes hybridized with DNA of ten C. psittaci strains and four C. trachomatis strains. These results indicated that the two fragments shared a DNA sequence common to the chlamydial genus.     

Rabbit autoantibodies to actin induced by immunization with modified homologous actins

Actin is a major antigen involved in the reaction of smooth muscle antibody positive sera from patients with chronic active hepatitis. In the present study, actin extracted from rabbit skeletal muscle was denatured by sodium dodecyl sulfate and was immunized into the rabbit, a homologous animal for actin. The rabbits, thus immunized, produced antibodies reactive with actins of homologous and heterologous animals. In addition, the antibodies showed reactivity with autologous actin. It indicates that the denatured homologous actin is capable of terminating immunological tolerance to actin and induces formation of autoantibody to rabbit actin. This phenomenon may be implicated in the occurrence of anti-actin antibody in sera from patients with chronic liver disease and several other diseases.     

Enzymatic isomerization and epimerization of D-erythrose 4-phosphate and its quantitative analysis by gas chromatography/mass spectrometry

An enzyme preparation from beef liver catalyzed the isomerization and epimerization of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate. The presence of D-erythrulose 4-phosphate and D-threose 4-phosphate was demonstrated by several analytical methods. After dephosphorylation, the presence of D-erythrulose and D-threose was confirmed by thin-layer chromatography, gas-liquid chromatography and an enzymatic method depending upon D-erythrulose reductase. The enzymatic products were also identified and simultaneously quantitated by a new procedure using gas chromatography/mass spectrometry. Each of three tetroses was distinguished by the combination of the reduction with sodium borodeuteride and the determination of relative intensities of the ion pairs m/z 379 and 380 of sugar tetritol trifluoroacetate. By gas chromatography/mass spectrometry, we observed that D-threose 4-phosphate was also converted into D-erythrulose 4-phosphate and D-erythrose 4-phosphate. At the equilibrium, about 90% of the tetrose 4-phosphate existed in the form of D-erythrulose 4-phosphate. On the basis of gas chromatography/mass spectrometric evidence together with gas chromatographic and thin-layer chromatographic patterns, it is suggested that the single enzyme of the beef liver catalyzed both reactions of isomerization and epimerization of aldotetrose 4-phosphate.     

Hypusine, N6-(4-amino-2-hydroxybutyl)-2,6-diaminohexanoic acid,in tissue proteins of mammals

Hypusine, N⁶-(4-amino-2-hydroxybutyl)-2,6-diaminohexanoic acid was isolated from proteins of bovine brain. Its identification was performed by comparison of its behavior in amino acid analysis, paper chromatography and electrophoresis to that of the authentic compound, and by periodic acid-permanganate oxidation which split hypusine into β-alanine and lysine. Hypusine was found in proteins of various organs of rabbits.Formation of hypusine from lysine was demonstrated by the intraperitoneal injection of labeled lysine into a rat and isolation of radioactive hypusine from the animal proteins. This findings indicates a possibility that hypusine is derived from the lysine residue of proteins through attachment of the 4-amino-2-hydroxybutyl moiety to the N⁶-amino radical of lysine.     

Myristoylation of neutrophil proteins and their biological characteristics

We have studied protein acylation in neutrophils of guinea pigs using [3H]myristate. A large number of neutrophil proteins were acylated with exogenously added myristic acid. The myristoylation was detected on 110, 77, 56, 54, 52, 42, and 37 kDa proteins. These myristoylations were stronger in peripheral blood than in peritoneal cells. Myristic acid was found to be covalently linked by an amid bond to these proteins since the proteins were resistant to boiling, chloroform/methanol and hydroxylamine treatment. Most myristoylated proteins appeared to be associated with the membrane fraction, while some of the proteins such as 77 kDa one was distributed also in the cytoplasm and translocated from the cytoplasm to the plasma membrane by stimulation. Lysozyme was myristoylated in vitro by the N-hydroxysuccinimide ester of myristic acid. The myristoylated lysozyme had an ability to be associated with phospholipid liposomes, and the membrane-associated lysozyme became a substrate of the rat brain Ca2+- and phospholipid dependent protein kinase (protein kinase C). These results indicate that myristoylation in neutrophil proteins may have an important role in metabolic regulation through their membrane association.     

Glycerol oxidase, a novel copper hemoprotein from Aspergillus japonicus. Molecular and catalytic properties of the enzyme and its application to the analysis of serum triglycerides

Glycerol oxidase purified from Aspergillus japonicus AT 008 had Mr = 400,000 and contained 1 mol of protoheme IX and 2 g atoms of copper/mol of enzyme protein. The absorption maxima of the oxidized form were found at 557, 530, 420, 280, and 238 nm, and those of the reduced form at 557 and 430 nm. Anaerobic addition of glycerol to the enzyme produced both a shift of the Soret band from 420 to 410 nm and bleaching of the alpha and beta bands at 557 and 530 nm. The ESR spectrum of glycerol oxidase showed three major signals at g = 1.99, g = 2.00, and g = 2.02. The signals at g = 1.99 and g = 2.02 were diminished by the anaerobic addition of glycerol, and the three signals completely disappeared after the addition of either dithionite or diethyldithiocarbamate. Exposure of glycerol oxidase to a borate buffer of pH 10.0 resulted in activation of the enzyme with concomitant enhancement of the ESR signals at g = 1.99 and g = 2.02. Since glycerol oxidase acts predominantly on glycerol, the enzyme can be employed in a specific colorimetric assay for serum triglycerides in combination with lipoprotein lipase.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The sequence of histological changes in the regional lymph node and other lymphoid organs of mice injected with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) or bacterial lipopolysaccharide (LPS) was followed. Injection of CPS-K, but not LPS, induced the following characteristic histological changes in the regional lymph node. In the early stage there was a marked decrease in the number of small lymphocytes, accompanied by the appearance of scattered fragmented nuclei and infiltration of polymorphonuclear neutrophilic leukocytes, and in the late stage there was marked proliferation of macrophage-like cells and pyroninophilic cells. Histological changes in the thymus and spleen and changes in cell populations in the bone marrow and peripheral blood after CPS-K injection were essentially the same as after LPS injection. Since CPS-K has a much stronger adjuvant action on antibody response than does LPS, it is suggested that the characteristic histological changes in the regional lymph node after injection of CPS-K are closely related to its extraordinarily strong adjuvant action.