Calcium-dependent association of annexin VI, protein kinase C alpha, and neurocalcin alpha on the raft fraction derived from the synaptic plasma membrane of rat brain.

A membrane microdomain enriched in cholesterol and sphingolipids or so called "raft" region was found to contain many signal transducing proteins such as GPI-anchored proteins, trimeric G proteins and protein tyrosine kinases. Because brain-derived raft contains two calmodulin-binding proteins, GAP-43 and NAP-22 as the major protein components, the raft domain is assumed to be important in the Ca(2+)-signaling. In this study, we analyzed protein components showing Ca(2+)-dependent binding to the raft of synaptic plasma membrane from rat brain. SDS-PAGE analysis of the protein components in the EGTA eluate from the raft prepared in the presence of Ca(2+)-ions showed the elution of 80 kDa, 68 kDa, 22 kDa, and 21 kDa proteins. These proteins were identified as protein kinase C alpha (80 kDa) and annexin VI (68 kDa) from the partial amino-acid sequencing, and neurocalcin alpha (22 kDa) and calmodulin (21 kDa) with western blotting and electrophoretic mobilities in the presence or absence of Ca(2+) ions. Further immunoblotting experiments showed the Ca(2+)-dependent association of conventional, but not non-conventional, subtypes of PKC to the raft.

Physiological expression of the 2',5'-oligoadenylate synthetase gene in mouse intestine.

2',5'-Oligoadenylate synthetase (2-5A synthetase) is an enzyme induced by interferon (IFN) that is considered to play an important role in IFN action. The normal mouse, not treated exogenously with any IFN or IFN inducers, has been shown to have an enhanced level of 2-5A synthetase activity, which is distributed among several lymphoid tissues. In the present report, we investigated the expression of the 42-kD 2-5A synthetase mRNA in the organs of normal mice using RNA blot hybridization and reverse transcriptase polymerase chain reaction (RT-PCR). Among the organs tested, intestinal tissues had the highest levels of this mRNA. Furthermore, the 42-kD 2-5A synthetase mRNA was expressed in intestines from germ-free mice and fetuses. Immunoblotting analysis using a monoclonal antibody against the 42-kD 2-5A synthetase revealed that the 42-kD enzyme as well as a cross-reactive protein of 30 kD were produced in the organs of the normal mouse.

Serum 2',5'-oligoadenylate synthetase activity during interferon treatment of chronic hepatitis B.

We measured 2',5'-oligoadenylate synthetase activities in serum and peripheral blood mononuclear cells from 10 patients with chronic hepatitis B who were being treated with interferon so as to determine whether 2',5'-oligoadenylate synthetase activity in serum reflected 2',5'-oligoadenylate synthetase activity in peripheral blood mononuclear cells, and whether it could be used to monitor interferon treatment. Pretreatment values of 2',5'-oligoadenylate synthetase activity in patients' serum and peripheral blood mononuclear cells were not statistically different from values from control subjects. When interferon was administered, serum levels of 2',5'-oligoadenylate synthetase began to rise within 3 hr, reached peak values at 12 hr and then declined. The levels of 2',5'-oligoadenylate synthetase activity both in serum and peripheral blood mononuclear cells increased substantially during interferon treatment, ranging 2- to 50-fold greater than initial levels. The levels of 2',5'-oligoadenylate synthetase in serum correlated closely with levels in peripheral blood mononuclear cells. In addition, when the levels of 2',5'-oligoadenylate synthetase rose during interferon administration, serum hepatitis B virus DNA polymerase values fell, and, in some cases, DNA polymerase rose again when 2',5'-oligoadenylate synthetase fell after discontinuation of interferon. These findings suggest that 2',5'-oligoadenylate synthetase activity in serum accurately reflects the antiviral effect of interferon and could be used to monitor interferon treatment.

Detection of elevated levels of 2-5A synthetase in serum from children with various infectious diseases.

By a sensitive radioimmunoassay method, (2'-5')oligoadenylate synthetase was detected in serum from patients with viral, bacterial, or mycoplasmal infections at elevated levels compared with enzyme levels in serum from healthy individuals and patients suffering from noninfectious diseases.

(2'-5') oligoadenylate synthetase in chicken embryo erythrocytes and immature red blood cells.

The appearance and induction of (2'-5')oligoadenylate synthetase (2-5A synthetase) in chicken embryo erythrocytes during development, and the activity and molecular size of this enzyme in immature red blood cells from anemic chickens were studied. Enzyme activity first appeared in the embryos on the 15th day of incubation, a marked increase being seen 1 or 2 days after hatching. In erythrocytes from early embryos without 2-5A synthetase activity, chicken interferon (5 IU/ml at most) induced the production of a large amount of the enzyme. In immature red blood cells from anemic chickens, only a small amount of 2-5A synthetase was detected in the nuclear fraction. The cytoplasmic fraction contained the smaller enzyme (about 45 kilodaltons), but the larger enzyme (85-120 kilodaltons) was scarcely detected in either fraction. The larger enzyme may be synthesized during the maturation of red blood cells.

Presence of (2'-5')oligoadenylate synthetase in avian erythrocytes.

(2'-5')Oligoadenylate synthetase (2-5A synthetase) was found in avian erythrocyte lysates from chicken, goose, and pigeon, with high levels being observed in chicken erythrocytes. No activities, however, were detected in erythrocytes from human, sheep, mouse, turtle, frog, trout, or lamprey. In chicken erythrocyte lysate, about 70% of ATP was converted to 2-5A molecules during a 20-h incubation, in which the tri- and tetra-adenylate were the major products. The tri-, tetra-, penta-, and hepta-adenylate were synthesized sequentially, but the levels of the di-adenylate were low throughout the reaction. 2-5A synthetase was also seen in erythrocytes from specific pathogen-free chickens, suggesting that the enzyme was not produced as a result of microbial infections. 2-5A synthetases from avian erythrocytes of chicken and pigeon were found not only in cytoplasms, but also in nuclei. No enzyme activity, however, was detected in the nuclear fraction of goose erythrocytes. The molecular size of 2-5A synthetase in nuclei from chicken erythrocytes was 45,000-60,000 daltons, while cytoplasms contained an 85,000- to 120,000-dalton enzyme. In addition, the synthetase was present in several types of chicken tissue including liver, intestine, bone marrow, spleen, bursa, pancreas, and thymus, but not in brain, heart, or stomach.

Immunological cross-reactivity between large and small (2'-5')oligoadenylate synthetases from pig cells.

Using glycerol gradient centrifugation, the molecular sizes of porcine (2'-5')oligoadenylate synthetases (2-5A synthetases) were estimated. The 2-5A synthetase purified from pig spleen was about 150 kDa, while the enzyme extracted from nuclei of Newcastle disease virus-infected pig epithelial cells (SK-h) was about 20-40 kDa. The nuclear 2-5A synthetase was selectively adsorbed to Protein A-Sepharose beads conjugated with anti-spleen 2-5A synthetase antibody. Thus, the smaller 2-5A synthetase in nuclei of pig cells shares a protein structure with the larger enzyme from pig spleen.

Relaxation effect of chloramphenicol on the stringent control in Escherichia coli.

In 10B601 (rel+) strain possessing a temperature-sensitive valyl-tRNA synthetase, chloramphenicol prevented the formation of guanosine-3'-diphosphate-5'-diphosphate (ppGpp) as well as the stringent control of stable RNA synthesis, under the conditions where the incorporation of valine into protein was still detectable i.e. at the lower restrictive temperatures. On the other hand, the effect of chloramphenicol was not observed at higher restrictive temperatures above 42 degrees C where the incorporation of valine was completely absent. Pretreatment of 10B601 cells with chloramphenicol before transfer to a high restrictive temperature (43.5 degrees C) did retard the onset of accumulation of ppGpp after the shift-up. Duration of the lag period was dependent on the concentration of chloramphenicol added. In parallel with the inability of the cells to accumulate ppGpp, stable RNA synthesis was permitted to continue at that high temperature. These results suggest that chloramphenicol traps aminoacyl-tRNA at the A-sites of ribosomes by damming-up the small flow of aminoacyl-tRNA under the restrictive supply of amino acids. Unchanged tRNA which has been located at the A-site is replaced by the charged one, thus resulting in the suppression of ppGpp formation and in the restoration of stable RNA synthesis.

Regulation of stable RNA synthesis and ppGpp levels in growing cells of Escherichia coli.

Under the balanced condition of growth of E. coli cells, no distinct difference is observed in stable RNA and protein synthesis between CP78 (rel+) and CP79 (rel minus), whereas a considerable difference is present in RNA accumulation between NF161 (rel+) and NF162 (rel minus), where NF161 smaller than NF162. The RNA content of NF161 is lower than that of NF162 in four different cultures with different growth rates. These two sets of isogenic pairs of rel+ and rel minus strains are commonly used in the study of rel gene function; however, NF161 is a mutant in the spoT gene whose product may be responsible for the degradation of ppGpp. The basal levels of ppGpp in these four strains growing with three different growth rates were examined: NF161 (rel+ spoT minus) has a much higher content of ppGpp than do other strains. Furthermore, the contents of ppGpp tend to be lower when the above four strains are growing at a faster rate. Thus a close correlation seems to exist between the content of RNA and the basal level of ppGpp under the condition of balanced growth.