Inactivation of the ATP-dependent DNase of Escherichia coli After Infection with Double-Stranded DNA Phages

The ATP-dependent DNase activity of Escherichia coli disappeared or was markedly reduced after infection with double-stranded DNA phages, T2, T3, T4, T5, T6, T7, lambda, phi80, and P1, but not with the single-stranded DNA phage f1, or the RNA phage Qbeta. This DNase activity was not reduced when chloramphenicol was added prior to phage infection.     

Inhibition of Cell Division and DNA Synthesis by Gibberellin in Isolated Zinnia Mesophyll Cells

A culture system of isolated mesophyll cells of Zinnia eleganswas used to examine the action of gibberellic acid (GA) on celldivision. Isolated Zinnia mesophyll cells cultured in a mediumcontaining auxin and cytokinin reinitiated cell division ina partly synchronized manner. When mesophyll cells isolatedfrom 21-day-old seedlings were used, GA added to the culturemedium at concentrations of 1 x 10^–6 M or higher suppressedthe initial rise in the number of divided cells. Tracer experimentswith [³H]-dThd revealed that GA treatment inhibited the incorporationof [³H]-dThd into DNA in the nucleus without inhibiting theuptake of [³H]-dThd into the cells, indicating that GA inhibitedDNA synthesis. GA applied at 48 h inhibited the incorporationof [³H]-dThd into DNA during the following 24 h, but GA appliedat 72 h did not inhibit the incorporation during the subsequent24 h. This suggests that GA affects the process of reinitiationof DNA synthesis, but does not affect DNA synthesis once cellshave become proliferative. (Received January 14, 1986; Accepted March 31, 1986)     

Expression of rat alpha 2-macroglobulin gene during pregnancy

Rat alpha 2-macroglobulin (alpha 2M) is a typical acute phase protein, the concentration of which in serum increases more than 100-fold after inflammation. It is also known that the protein increases during pregnant (and neonatal) stages. Using a specific cDNA probe, expression of the alpha 2M gene during pregnancy was studied at the mRNA level. During inflammation, the liver is almost the only organ producing alpha 2M, but during pregnancy the placenta and uterus were found to be major organs producing a large amount (70-80% of that of inflamed liver) of alpha 2M mRNA at days 12-15. The yolk sac, maternal liver and fetal (or neonatal) liver also produced a small but significant amount (5-20% of that of inflamed liver) of the mRNA. Southern blotting analysis showed that only one copy of the alpha 2M gene was present in a haploid rat genome. These results indicated that a single alpha 2M gene has the ability to respond to two completely-different physiological states.     

Two types of coagulogen mRNAs found in horseshoe crab (Tachypleus tridentatus) hemocytes: molecular cloning and nucleotide sequences

The complete cDNA sequence coding for the coagulogen present in the horseshoe crab (Tachypleus tridentatus) hemocytes was determined. Clones carrying cDNA fragments for coagulogen were isolated from a cDNA library of the hemocyte mRNA using synthetic oligodeoxyribonucleotides as probes. The nucleotide sequence analyses of the cloned cDNAs revealed that the hemocyte coagulogen consists of 175 amino acids with 20 amino acids in a presegment, and that there are two types of mRNAs for coagulogen. The two mRNAs exhibited three nucleotide substitutions, two of which were in their protein-coding regions, resulting in two amino acid replacements. Subsequently, two molecular species of coagulogen, named coagulogens type I and type II, were identified by tryptic peptide mapping of the mature proteins isolated from the hemocyte lysate. These results suggest that the two types of coagulogens are first synthesized as preproteins and are incorporated into the granules that are abundantly present in the hemocytes with liberation of the signal peptides.     

Purification and characterization of the Ca2+ plus Mg2+-dependent endodeoxyribonuclease from calf thymus chromatin

Ca2+ plus Mg2+-dependent endodeoxyribonuclease was extracted from calf thymus chromatin and purified to a state free from contamination by other DNases. This DNase required both Ca2+ and Mg2+, or Mn2+ alone for its activity and the optimum pH for activity was at 6.5-7.5. No specificity for the 5'-base was observed. The molecular weight of the DNase was estimated to be about 25,000-30,000 by glycerol gradient centrifugation. Actin and antibody for pancreatic DNase (DNase I) did not inhibit the enzyme, whereas both strongly inhibited DNase I, suggesting that these two DNases are different enzymes.     

Cytochrome b558, a component of the phagocyte NADPH oxidase, is a flavoprotein.

Cytochrome b558 is the only membrane component of the phagocyte O2(-)-producing NADPH oxidase. The O2- production by the oxidase reconstituted in vitro with the crude membrane fraction is enhanced several-fold by addition of FAD, whereas that with the partially purified cytochrome is completely dependent on exogenous FAD, suggesting that FAD acts through the membrane component, cytochrome b558. The alignments of the amino acid sequence of the large subunit of the cytochrome (gp91-phox) with those of previously characterized flavoproteins reveal that the middle and C-terminal portions of gp91-phox are likely to be FAD- and NADPH-binding domains, respectively. Cytochrome b558, thus, appears to be a flavoprotein with an NADPH-binding site, of the NADPH oxidase.     

Diagnosis of familial amyloidotic polyneuropathy by recombinant DNA techniques

A calmodulin dependent cyclic nucleotide phosphodiesterase is associated with the head and tailpieces of demembranated rat caudal epididymal sperm. The phosphodiesterase was stimulated two-fold in the presence of Ca2+, while the simultaneous addition of Ca2+ and calmodulin resulted in a four-fold increase in activity. Ca2+ stimulation was abolished if demembranated sperm were extracted with EGTA and was recovered upon the addition of exogenous calmodulin. Micromolar levels of Ca2+ were required for full stimulation. Trifluoperazine inhibited the Ca2+ stimulated enzyme in a dose dependent manner (ID50 = 50 microM) but had no effect on the basal phosphodiesterase activity.     

Spin-label studies of the oligomeric structure of band 3 protein in erythrocyte membranes and in reconstituted systems

A spin-labeled fatty acid (16-doxylstearic acid), linked by an ester bond to a maleimide or a nitrene residue, was covalently attached to band 3 of erythrocyte membranes. The electron spin resonance spectrum of the spin-labeled protein was examined at different temperatures in: (a) whole erythrocyte ghosts; (b) ghosts depleted of spectrin and actin; (c) alkaline-treated ghosts; (d) vesicles made with purified band 3 reassociated with dimyristoylphosphatidylcholine. Most spectra are composite with a major component corresponding to a large overall splitting. The determination of the percentage of the immobilized component was carried out by pairwise subtraction. At low temperatures (1–7°C), the highest fraction of immobilized component was found in dimyristoylphosphatidylcholine vesicles (approx. 100%); alkaline-treated membranes had approx. 75% of the immobilized component at the same temperature; whole erythrocyte, spectrin/actin-depleted and spectrin/actin/ankyrin-depleted ghosts gave identical results (approx. 60% of immobilized component). The immobilized fraction decreased in all samples with increasing temperature or addition of a nonsolubilizing concentration of dodecyl octaethylene glycol monoether. In dimyristoylphosphatidylcholine vesicles, however, the modification in the ratio of the two components was obtained only above the lipid transition temperature (23°C). The strong immobilization of the spin-labeled lipid chain at all temperatures suggested trapping of the lipid chain between proteins. At low temperature, in dimyristoylphosphatidylcholine vesicles or in alkaline-treated ghosts, lipid-protein segregation is likely to take place. In whole erythrocyte ghosts, on the other hand, the large contribution of the motionally restricted component at physiological temperature indicates the oligomeric nature of band 3. Partial dissociation of the oligomers occurs as the temperature is increased, but the presence or absence of cytoskeletal proteins has no influence on the state of oligomerization of band 3.     

Subcellular distribution of the mannan-binding protein and its endogenous inhibitors in rat liver

The subcellular distribution of the mannan-binding protein from rat liver, a lectin specific for mannose and N-acetylglucosamine, was studied. Approximately 75% of the binding activity of the homogenate was recovered in microsomes, approximately 76% of which was accounted for by rough microsomes. Rough microsomes had the highest specific activity of binding, followed by the Golgi apparatus and smooth microsomes, whereas plasma membranes, lysosomes, mitochondria, and the soluble fraction had little or no binding activity. A topographical survey indicated that the binding protein was localized exclusively on the cisternal surface of microsomal vesicles. Thus, the binding protein of microsomal vesicles was protected from protease digestion and was released from the vesicles by mild detergent treatment. Competitive inhibitors, which presumably represent endogenous ligands of the binding protein, were found among subcellular fractions. More than 50% of the inhibitory activity of the homogenate was recovered in rough microsomes, while the highest specific activity of inhibition was found in lysosomes. The K_i values estimated for rough microsomes and lysosomes were 25.9 and 8.67 μg/ml, respectively. The distribution profiles of inhibitors were correlated roughly with those of the binding protein, resulting in masking of the binding activity in organelles up to the level of 86%. On the basis of the known localization and topology of the binding protein and endogenous inhibitors (ligands), possible physiological functions of the binding protein relevant to the transport of biosynthetic intermediates of glycoproteins from the rough endoplasmic reticulum to the Golgi apparatus and from the Golgi apparatus to lysosomes were discussed.