Localization of 3H-proline, 35S-sulfate and 3H-glucosamine in rat cornea following epithelial removal: epithelio-stromal interaction

Localization of 3H-proline, 35S-sulfate and 3H-glucosamine was studied by autoradiography in the rat cornea following the removal of the epithelium. The three labeling chemicals were injected into the anterior chamber of rats, one chemical in each rat, 24 hours after the removal of the epithelium. Animals were sacrificed at various intervals up to 7 days after the injection. The silver grains of 35S-sulfate incorporated into the re-covered epithelium gradually shifted into the stroma. On the other hand, the 3H-glucosamine tended to move toward the epithelial cell membrane and the superficial layer of the epithelium. The 3H-proline incorporated in the epithelium remained in the cells without movement. These results suggest that the 35S-sulfate in the epithelium shifted into the stroma as 35S-phosphoadenosine-phosphosulfate [35S-PAPS] before sulfation of glycoconjugates occurred in the epithelium. A large amount of 3H-glucosamine was utilized as a component of low-sulfated glycoconjugates in the epithelial cell membrane and other cell-coating substances.     

Stromal changes following removal of epithelium in rat cornea

Morphologic and metabolic changes were studied in the corneal stroma of the rat eye following denudation of the epithelium. By touching the cornea with a glass slide coated with 10% gelatin the epithelium was removed with minimal trauma to the underlying stroma. The underlying superficial keratocytes degenerated promptly leaving an anterior acellular zone within 12 hours. At 24 hours wandering cells invaded this damaged stroma, coincident with reepithelialization of the surface. This suggests not only an interaction of epithelium and keratocytes but also of epithelium and wandering cells. Repair of the acellular zone began about 24 hours after the denudation. At 48 hours mitotic figures were abundant with vigorous incorporation of the 3H-thymidine in the remaining keratocytes. The acellular zone became repopulated with new stromal cells advancing from the posterior stroma toward the anterior stroma.     

Inhibition of 2-nitropropane-induced rat liver DNA and RNA damage by benzyl selenocyanate

We observed that pretreatment of male F344 rats with benzyl selenocyanate, a versatile organoselenium chemopreventive agent in several animal model systems, decreases the levels of DNA and RNA modifications produced in the liver by the hepatocarcinogen 2- nitropropane. To clarify the mechanisms involved, we pretreated male F344 rats with either benzyl selenocyanate, its sulfur analog benzyl thiocyanate, phenobarbital or cobalt protoporphyrin IX; the latter is a depletor of P450. We then determined (1) the ability of liver microsomes to denitrify 2-nitropropane, (2) effects on 2-nitropropane- induced liver DNA and RNA modifications and (3) amount of nitrate excreted in rat urine following administration of the carcinogen. Pretreatment with benzyl selenocyanate or phenobarbital increased the denitrification activity of liver microsomes by 217 and 765%, respectively, increased liver P4502B1 by 31- and 435-fold, respectively, decreased the levels of 2-nitropropane-induced modifications in liver DNA (29-70% and 17-30%, respectively) and RNA (67-85% and 30-50%, respectively), and increased the 24-h urinary excretion of nitrate by 157 and 209%, respectively. Pretreatment with benzyl thiocyanate had no significant effect on any of these parameters. Pretreatment with cobalt protoporphyrin IX decreased liver P4502B 1 by 87%, decreased the denitrification activity of liver microsomes by 76%, decreased the 24 h urinary excretion of nitrate by 88.5%, but increased the extent of 2-nitropropane-induced liver nucleic acid modifications by 17-67%. These results indicate that the metabolic sequence from 2-nitropropane to the reactive species causing DNA and RNA modifications does not involve the removal of the nitro group. Moreover, they suggest that benzyl selenocyanate inhibits 2-NP-induced liver nucleic acid modifications in part by increasing its detoxication through induction of denitrification, although it is evident that other mechanisms must also be involved.      

Disseminated intravascular coagulation after hepatic resection

Disseminated intravascular coagulation (DIC) after hepatic resection is a serious complication that leads to a fatal outcome unless prompt treatment is instituted. Between April 1973 and June 1988, DIC occurred postoperatively in 18 of 192 patients who underwent hepatic resection because of a variety of diseases of the liver and biliary tract. The diagnosis was made on the basis of changes in platelet count, fibrinogen level, serum level of fibrin degradation product (FDP), and protamine sulfate test. Heparin was used in an earlier series but has been discontinued because of difficulty in determining the optimal dose in patients undergoing liver resection. Instead, we now use gabexate mesilate, which blocks the coagulation cascade without the aid of antithrombin III and works as an anticoagulant. Fifteen patients had uneventful recoveries, but three died. Two died of aggravation of DIC, which was a result of reoperation performed under the diagnosis of surgical bleeding. The other patient died of liver failure after fever of unknown cause persisted for 4 months. The rationale for the diagnosis and treatment of DIC after liver resection is documented, and the problems involved are discussed.     

Trypanosoma cruzi histone H1 is phosphorylated in a typical cyclin dependent kinase site accordingly to the cell cycle

Histone H1 of most eukaryotes is phosphorylated during the cell cycle progression and seems to play a role in the regulation of chromatin structure, affecting replication and chromosome condensation. In trypanosomatids, histone H1 lacks the globular domain and is shorter when compared with the histone of other eukaryotes. We have previously shown that in Trypanosoma cruzi, the agent of Chagas' disease, histone H1 is phosphorylated and this increases its dissociation from chromatin. Here, we demonstrate using mass spectrometry analysis that T. cruzi histone H1 is only phosphorylated at the serine 12 in the sequence SPKK, a typical cyclin-dependent kinase site. We also found a correlation between the phosphorylation state of histone H1 and the cell cycle. Hydroxyurea and lactacystin, which, respectively, arrest parasites at the G1/S and G2/M stages of the cell cycle, increased the level of histone H1 phosphorylation. Cyclin-dependent kinase-related enzymes TzCRK3, and less intensely the TzCRK1 were able to phosphorylate histone H1 in vitro. Histone H1 dephosphorylation was prevented by treating the parasites with okadaic acid but not with calyculin A. These findings suggest that T. cruzi histone H1 phosphorylation is promoted by cyclin dependent kinases, present during S through G2 phase of the cell cycle, and its dephosphorylation is promoted by specific phosphatases.