Novel gelatinolytic activities in rat organs.

Novel gelatinolytic activities in both latent and active forms were detected in the normal organs of rat by gelatin zymography. Multiple active bands were detected in the extracts from the skin, jejunum, muscle, and kidney without any activation. These activities were inhibited by 1,10-phenanthroline or leupeptin, nor by E64, suggesting that these activities were derived from metallo-proteinases or serine-proteinases. Some gelatinolytic active bands were newly induced or enhanced by p-aminophenylmercuric acetate. These results suggest that matrix degrading activities due to metallo- and serine-proteinases were constitutively expressed in various rat normal organs.     

Glycogen metabolizing enzyme activities in the developing rat liver

1. The glycogen content of rat liver increased prenatally and dramatically decreased after the birth. 2. Glycogen synthase and glycogen phosphorylase activities increased prenatally, declined at 12 hr after birth and then again increased. 3. Phosphorylase kinase activities did not significantly change prenatally but steadily increased after the birth. 4. Protein kinase activities (units/mg liver) did not change prenatally but slightly increased after the birth. 5. Phosphoprotein phosphatase activities using phosphorylase a and histone as substrates dramatically increased at birth and then decreased after 24 hr of the birth.     

Special cutaneous receptor organs of fish. IV. Ampullary organs of the nonelectric catfish, Kryptopterus

Ampullary organs of the transparent catfish, Kryptopterus bicirrhus, are present in large numbers on the head and in a regular pattern of lines on the body and fins. The organs lie in the epidermis, and have a pore that opens to the surface. Flattened cells form a roof and walls. On the floor of the organ there are a “sensory hillock,” composed of spherical receptor cells and columnar supporting cells, and a “secretory hillock” composed of columnar secretory cells. The receptor cells are nonciliated and have only afferent innervation. The organ cavity is filled with jelly. The organs are compared with ampullary organs of the weakly electric fish Eigenmannia, ampullae of Lorenzini of Raja, and small pit organs of Amiurus. Structural characteristics of the ampullary organs of Kryptopterus make them especially suitable for electrophysiological studies.     

Effect of thyroid hormones on the activities of hepatic alcohol metabolizing enzymes.

Treatment with thyroxine or triiodothyronine for 7 days in order to simulate a hyperthyroid state results in an enhanced activity of the microsomal ethanol oxidizing system. Conversely, a decrease of hepatic alcohol dehydrogenase activity was observed under these experimental conditions, whereas hepatic catalase activity remained unchanged. These findings suggest that if chronic ethanol consumption simulates a “hyperthyroid hepatic state”, increased rates of ethanol metabolism observed following prolonged alcohol intake might therefore be attributed at least in part to an induction of microsomal ethanol oxidizing system activity in the liver.     

Potential activities of androgen metabolizing enzymes in human prostate

The entire androgen metabolism of the human prostate is an integral part of the DHT mediated cellular processes, which eventually give rise to the androgen responsiveness of the prostate. Therefore, the potential activities of various androgen metabolizing enzymes were studied. Moreover, the impact of aging on the androgen metabolism and the inhibition of 5-reductase by finasteride were studied. In epithelium (E) and stroma (S) of normal (NPR) and hyperplastic human prostate (BPH), for each enzyme being involved in the conversion either of testosterone via DHT, 3- and 3β-diol to the C₁₉O₃-triols or from testosterone to androstenedione and vice versa, the amount (V_max) and Michaelis constant (K_m) were determined by Lineweaver-Burk plots. Furthermore, V_max/K_m quotients were calculated, which served as an index for the potential enzyme activity. 17 enzymes showed a mean V_max/K_m ≥ 0.10. The top four were the 5-reductases in E and S of NPR and BPH. Among those, the highest activity was found in E of NPR (1.6 ± 0.2). Moreover, in E a significant age-dependent decrease of 5-reductase activity occurred, whereas in stroma rather constant activities were found over the whole age range. Similar age-dependent alterations were found for the cellular DHT levels. Finally, the finasteride inhibition of 5-reductase (IC₅₀;nM) was stronger in E (35 ± 17) than in S (126 ± 15). In conclusion, 5-reductase is: (a) the outstanding androgen metabolizing enzyme in NPR and BPH; (b) dictating the DHT enrichment in the prostate; (c) under the impact of aging; and (d) preferentially inhibited by finasteride in E.     

Effects of structural modifications of N-CPM-normorphine derivatives on agonist and antagonist activities in isolated organs

The agonistic and antagonistic properties of N-cyclopropylmethyl (N-CPM) morphine derivatives were observed in mouse vas deferens (MVD), longitudinal muscle of guinea pig ileum (GPI) and rabbit vas deferens (LVD). In MVD the K(e) values of the titled compounds (N-CPM-morphine, N-CPM-isomorphine, N-CPM-dihydromorphine, N-CPM-dihydroisomorpPhine, N-CPM-dihydromorphone and naltrexone) were measured for mu-, kappa- and delta-receptors using normorphine, ethylketocyclazocine (EKC) and D-Pen2-D-Pen5-enkephaline (DPDPE) as selective agonists on the receptors, respectively. For mu-receptors of MVD the tested compounds showed similar affinity. For kappa-receptors the non-iso-6-OH derivatives possessed much less affinity than the iso-derivatives. Similar difference could be observed for delta-receptors. The agonistic activities of these compounds in MVD were observed to be between 0-20% of the inhibition of muscle contractions. In GPI the compounds except naltrexone possessed strong agonistic activities effectively antagonized by nor-binaltorphimine (nor-BNI) (K(e) of nor-BNI was 0.23 nM) suggesting that they were strong kappa-receptor agonists. We investigated these agents in LVD too, which contains kappa-receptors, but they did not produce any agonist potencies. It raises the possibility that the kappa-receptor subtypes of LVD and MVD are different from the kappa-receptor subtype of GPI or the vasa deferentia contain much fewer kappa-receptors than GPI and the intrinsic activities of these compounds are too small to reach the 50% inhibition of the contractions.     

Chronic lithium treatment prevents the dexamethasone-induced increase of brain polyamine metabolizing enzymes.

The paper describes the effects of various regimens of lithium chloride treatment on dexamethasone-induced increases in brain polyamine metabolizing enzymes. In contrast to peripheral tissues where acute lithium treatment suppresses the increase in ornithine decarboxylase activity, in the brain only chronic treatment was effective in preventing this increase and also the increases in the activities of S-adenosylmethionine decarboxylase and spermidine/spermine N1-acetyltransferase. This findings indicate a novel brain target for lithium's action and in turn provide new avenues for exploring polyamine function in the brain.     

Diurnal variations in the activities of the glycogen metabolizing enzymes in mouse liver

The glycogen level in mouse liver was maximal during the night and decreased to the lowest level during the light period. The peak activity of phosphorylase alpha was observed during the light hours and thus paralleled the decline of hepatic glycogen concentrations. The period of rapid glycogen synthesis (1800-2200 hr) was immediately preceded by maximum glycogen synthase alpha activity. Significant diurnal rhythms for phosphorylase kinase and phosphorylase phosphatase activities were also observed and appear to play a role in regulating the diurnal rhythm of phosphorylase alpha activity.     

Characterization of DNA metabolizing enzymes in situ following polyacrylamide gel electrophoresis

We have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure. DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined 32P-labeled oligonucleotides annealed to M13 DNA. After electrophoresis, these enzymes catalyzed in situ reactions and their [32P]DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel. Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities. The catalytic and physical properties of Novikoff hepatoma DNA polymerase beta were found to be similar under both in vitro and in situ conditions. With 3'-terminally matched and mismatched [32P]DNA substrates in the same activity gel, DNA polymerase and/or 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized. In addition, use of matched and mismatched DNA primers permitted the uncoupling of mismatch excision and chain extension steps. Activities first detected in nondenaturing activity gels as either multifunctional or multimeric enzymes were also identified in denaturing activity gels, and assignment of activities to specific polypeptides suggested subunit composition. Furthermore, DNA substrates cast within polyacrylamide gels were successfully modified by the exogenous enzymes polynucleotide kinase and alkaline phosphatase before and after in situ detection of E. coli DNA ligase activity, respectively. Several restriction endonucleases and the tripeptide (Lys-Trp-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA. This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways.     

Drug metabolizing enzymes in cerebrovascular endothelial cells afford a metabolic protection to the brain.

The brain is partially protected from chemical insults by a physical barrier mainly formed by the cerebral microvasculature, which prevents penetration of hydrophilic molecules in the cerebral extracellular space. This results from the presence of tight junctions joining endothelial cells, and from a low transcytotic activity in endothelial cells, inducing selective permeability properties of cerebral microvessels that characterize the blood-brain barrier. The endothelial cells provide also, as a result of their drug-metabolizing enzymes activities, a metabolic barrier against potentially penetrating lipophilic substances. It has been established that in cerebrovascular endothelial cells, several families of enzymes metabolize potentially toxic lipophilic substrates from both endogenous and exogenous origin to polar metabolites, which may not be able to penetrate further across the blood-brain barrier. Enzymes of drug metabolism present at brain interfaces devoid of blood-brain barrier, like circumventricular organs, pineal gland, and hypophysis, that are potential sites of entry for xenobiotics, display higher activities than in cerebrovascular endothelial cells, and conjugation activities are very high in the choroid plexus. Finally, xenobiotic metabolism normally results in detoxication, but also in some cases in the formation of pharmacologically active or neurotoxic products, possibly altering some blood-brain barrier properties.     

The many consequences of chemical- and genetic-based modulation of drug metabolizing enzyme activities.

The induction or inhibition of the metabolizing enzyme activities by a great deal of substances (including drugs) influence their toxicological or pharmacological outcomes as well as that of other xenobiotics or drugs to which human is simultaneously exposed. The dual bioactivating/detoxificating nature of both phase I and phase II enzymes poses such modulation as an unavoidable unhealthy phenomenon. Therefore, the proposed strategies in preventive medicine which foresee boosting or depressing enzymatic effects such as those in the field of cancer chemoprevention, should be carefully reconsidered before their credibility would be compromised. As the phenotypic features, genetic polymorphisms leading to the occurrence of high or low metabolizers in the population, each at high risk to certain forms of toxicity, behave as a sort of "constitutive" enzymatic modulation. Thus, considering the double-edged sword nature (detoxi-toxicant) of these catalysts towards ubiquitous environmental pollutants, the search for individual susceptibility by means of the genotypic analysis represents a very intriguing problem. However, the knowledge of the "overall" metabolic fingerprint associated to the phenotypic analysis in a single person could offer an interesting way to (partially) control human risk by making suitable (well aimed) modifications of determined life-styles (e.g. stop smoking or drinking) or particular dietetic practices (e.g. stop eating high cooked meat or fish) as well as selecting personalised drug adjustments by physicians either in terms of dosage or fitting drug.