Hepatic drug-metabolizing enzyme system and endotoxin tolerance: structural requirement of LPS in induction of an early tolerance

The alteration of hepatic drug-metabolizing enzyme activities in mice given Salmonella endotoxin by single or multiple intraperitoneal injections was investigated. An essentially the same biphasic, early and late phase, endotoxin tolerance was observed in the animals receiving a single injection of endotoxin or repetitive daily injections. The results of reciprocal cross tolerance tests using lipopolysaccharide and free lipid A preparations derived from Salmonella minnesota, Salmonella typhimurium, E. coli, Pseudomonas aeruginosa, and Chromobacterium violaceum suggested that lipid A moiety plays an important role in the induction of early endotoxin tolerance to endotoxin response.     

Phosphorylation and Inactivation of Brain Glycogen Synthase by a Multifunctional Calmodulin-Dependent Protein Kinase

Glycogen synthase was partially purified from canine brain to about 70% purity. The purified enzyme showed differences from the properties of the skeletal muscle enzyme with respect to molecular weights of the holoenzyme and subunit and phosphopeptide mapping. The multifunctional calmodulin-dependent protein kinase from the brain phosphorylated brain glycogen synthase with concomitant inactivation of the enzyme. Although about 1.3 mol of phosphate/mol subunit was maximally incorporated into glycogen synthase, 0.4 mol of phosphate/mol subunit was sufficient for the maximal inactivation of the enzyme. The results indicate that brain glycogen synthase is regulated in a calmodulin-dependent manner similarly to the skeletal muscle enzyme, but that the brain enzyme is different from the skeletal muscle enzyme.     

Electric charge distribution in proteins and ionic strength dependence of reaction rate

The ionic strength dependence of the reaction rate between protein and dichloride anion radical has been investigated by flash photolysis of aqueous chloride-containing lysozyme, ribonuclease A, or insulin. The rate constant for the reaction of lysozyme or ribonuclease A with dichloride anion radicals decreases with increasing ionic strength, while it increases for insulin. The dependence was found to obey an equation derived from the theory of Debye and Hückel or the equation of Wherland and Gray for lysozyme within experimental errors. For ribonuclease A, however, it deviates largely from these equations. In the case of insulin a moderate deviation was observed. The different behavior in the ionic strength dependence is discussed in terms of the electric charge distribution in the protein molecules.     

Nitric Oxide Reversibly Suppresses Xanthine Oxidase Activity

The effects of nitric oxide (NO) on xanthine oxidase (XOD) activity and the site(s) of the redox center(s) affected were investigated. XOD activity was determined by superoxide (O₂^-) generation and uric acid formation. NO reversibly and dose-dependently suppressed XOD activity in both determination methods. The suppression interval also disclosed a dose-dependent prolongation. The suppression occurred irrespective of the presence or absence of xanthine; indicating that the reaction product of NO and O₂^-, peroxynitrite, is not responsible for the suppression. Application of synthesized peroxynitrite did not affect XOD activity up to 2 μM. Methylene blue, which is an electron acceptor from Fe/S center, prevented the NO-induced inactivation. The results indicate that NO suppresses XOD activity through reversible alteration of the flavin prosthetic site.     

Superoxide Scavenging Activity of Spin-Labeled Nitrosourea and Triazene Derivatives

Superoxide scavenging activities (SSA) of newly synthesized spin-labeled nitrosourea and triazene derivatives, and their precursor nitroxides were investigated by the ESR/spin-trapping method using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and hypoxanthine/xanthine oxidase as the superoxide-generating system. The spin-labeled nitrosoureas, triazenes and their precursor nitroxides exhibited excellent SSA, whereas clinically used nitrosourea and triazene, which do not contain the nitroxide moiety, did not show any SSA. Furthermore, it was deduced that these nitroxides scavenge superoxide by redox cycling between nitroxide and corresponding hydroxylamine.     

Cell Sorting and Chondrogenic Aggregate Formation in Limb Bud Recombinants and in Culture

The cartilage pattern of the developing chick limb changes along the proximal-distal (PD) axis. It is assumed that these spatial changes are brought about by differences in the cellular properties of distal mesoderm, the progress zone (PZ). To examine whether these differences are actually maintained in the individual cells composing the PZ, we dissociated early (stage 20) and late (stage 25) PZ tissues into single cells, then mixed and recombined them with ectodermal jackets. The recombinants were grafted to limb bud stumps and allowed to develop into limb-like structures. Early PZ cells were distributed within whole cartilage elements along the PD axis of the limb-like structures, while cells from late PZ participated only in the formation of distal cartilage elements.
A difference in distribution pattern between the cells of early and late PZ in mixed culture was also observed. Cells of early PZ aggregated rapidly in patches and formed cartilage nodules, while the cells of late PZ distributed in regions surrounding these cell aggregates and gradually differentiated to cartilage cells. These results suggest that the cellular properties in the PZ concerning the rate of chondrogenic aggregate formation change during limb bud development, and that this change may relate to the cartilage pattern formation along the PD axis.     

Expression of listeriolysin O by intracellular Listeria monocytogenes following infection of lipopolysaccharide-treated or untreated J774.1 macrophage-like cells

Abstract Listeria monocytogenes replicates in a phagocytic cell following escape into the host cytoplasm. Listeriolysin O, secreted by L. monocytogenes , which belongs to the thiol-activated hemolysin family, is known to play an important role in the escape of the bacterium into the host cytoplasm. In this study, we demonstrated that expression of listeriolysin O by infecting L. monocytogenes was lightly induced in J774.1 macrophage-like cells pretreated with lipopolysaccharide, although the growth of the bacteria was suppressed. The number of viable L. monocytogenes decreased until 4 h post-infection and then increased between 4 and 8 h post-infection in untreated J774.1 host cells, but it decreased until 8 h post-infection in lipopolysaccharide-treated host cells. However, expression of listeriolysin O by L. monocytogenes was not induced in the untreated host cells, while it increased between 0 and 4 h post-infection in the lipopolysaccharide-treated host cells. Expression of listeriolysin O mRNA in the lipopolysaccharide-treated host cells was also induced at 2 h post-infection, suggesting that listeriolysin O was newly synthesized in the macrophage-like cells. These results suggest that macrophage activation induced with lipopolysaccharide could lead to the expression of the listeriolysin O gene and the synthesis of listeriolysin O protein under suppression of the intracellular growth of L. monocytogenes .     

Prevention of Vascular Graft Infection by Sisomicin Incorporated into Fibrin Glue

To examine the efficacy of sisomicin (SISO) incorporated into fibrin glue (FG) for the prevention of graft infection in animal models, the susceptibility to infection of Dacron grafts (control) and SISO-FG Dacron grafts following the inoculation of Staphylococcus aureus or S. epidermidis was compared. The results showed that SISO-FG Dacron grafts displayed resistance to graft infection.     

CDP-diglyceride:inositol transferase from rat liver. Purification and properties.

CDP-diglyceride:inositol transferase, which catalyzes the final step of the de novo synthesis of phosphatidylinositol, was solubilized by sodium cholate from microsomes prepared from rat liver and purified by ammonium sulfate fractionation, sucrose density gradient centrifugation, and DEAE-cellulose column chromatography. Addition of phospholipid during the purification and the assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation was nearly homogeneous as judged by polyacrylamide gel electrophoresis. The recovery of the purified enzyme from the microsomal fraction was 3 to 3.3% with respect to activity and 0.12% with respect to amount of protein. The molecular weight of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 60,000. The purified enzyme required exogenous phospholipds for its activity. Various phospholipid classes activated the enzyme rather nonspecifically. The Km for myo-inositol was 2.5 X 10(-3) M and that for CDP-diglyceride was 1.7 X 10(-4) M. The pH optimum was 8.6. The enzyme required Mm2+ or Mg2+ for activity. The optimal concentration of Mn2+ for activation was 0.5 mM, while the activity in the presence of Mg2+ increased up to 20 mM. The enzyme was inhibited by thiol-reactive reagents. There was a competition for inositol by inosose-2 but not by scyllitol.     

Control of the production of orthophosphate repressible extracellular enzymes in Neurospora crassa

Molecular Genetics and Genomics - The abilities of purine- and pyrimidinerequiring mutants to produce six orthophosphate repressible extracellular enzymes, alkaline phosphatase,...