Decrease in hepatic drug-metabolizing enzyme activities after removal of rats from pine bedding

Wood is often used as a contact bedding material for laboratory animals. It has been established that wood, particularly softwood, has the potential to induce hepatic drug-metabolizing enzymes. However, to the authors' knowledge, changes in enzyme activity after removal of animals from bedding have not been characterized. The purpose of the study reported here was to determine how hepatic ethoxyresorufin-O-deethylation and p-nitrophenol hydroxylation activities alter over time after removal of rats from pine bedding. Male rats, three to four months old, raised in cages containing pine bedding, were transferred to wire-bottomed cages. At various times thereafter (up to 84 days), groups of rats were euthanized and the liver was processed to obtain microsomes. The microsomal protein and total cytochrome P450 (CYP) content and enzyme activities were determined. Significant differences in total microsomal protein or total CYP values were not observed over the 84 days, but a decrease in ethoxyresorufin-O-deethylation and p-nitrophenol hydroxylation activities was detected. For p-nitrophenol hydroxylation, the decrease was exponential, with a half-life of approximately nine days, whereas for ethoxyresorufin-O-deethylation, a rapid decrease in activity in the first week was followed by a reduced rate of decrease thereafter. Enzyme activities did not stabilize for at least six weeks. Researchers using laboratory animals should, therefore, be aware that it takes several weeks for enzyme activities to stabilize once animals are removed from the bedding.     

Response of the rat's hepatic drug-metabolizing enzyme system to chemotherapy of Trypanosoma b. brucei infections with Berenil and Suramin

Berenil (4,4-diamidinodiazoaminobenzene-diacetamide acetate) or Suramin [sodium salt of 8-(3-benzamido-4-methylbenzamido)-naphthalene-1,3,5-trisulfonic acid] treatment of rats infected with Trypanosoma b. brucei enhanced hepatic microsomal aniline hydroxylase and p-aminopyrine N-demethylase activities. While Suramin inhibited significantly the activities of cytoplasmic glutamate dehydrogenase and lactate dehydrogenase, Berenil had no effect. The kinetic profiles of these enzymes consistently showed a Km value similar to that of controls. Both cytosolic and microsomal glutathione-S transferase and microsomal epoxide hydratase were unaffected by Suramin. However, a significant increase in cytosolic glutathione-S transferase was observed with Berenil. Microsomal phospholipids were not affected by any of the drugs.     

Effect of herb supplement on hepatic enzyme activities in ddY mice.

Plasma glucose and lipid concentrations and hepatic enzyme activities were measured in male ddY mice supplemented with the herb, Echevaria glauca, to examine the effect of herbal treatment. In mice supplemented with the herb, plasma triglyceride (TG) and free fatty acid (FFA) concentrations decreased and hepatic glycolytic enzyme and glutathione peroxidase (GSHpx) activities increased significantly compared with those in the non-treated control mice. These increases in hepatic enzyme activities were not fully dose-dependent, however the higher dose and longer duration with herb supplement induced increases in the enzyme activities. It was found that dietary herb supplement caused an acceleration of hepatic function, judged by increased activities of glycolytic enzyme and GSHpx in ddY mice.     

Mycolic acid-containing glycolipid as a possible virulence factor of Rhodococcus equi for mice

By the use of various Rhodococcus equi strains differing in the length of carbon chains of glycolipid, we examined whether the glycolipid, glucose monomycolate, was contributing to the virulence of R. equi for mice. R. equi strains with longer carbon chain mycolic acid showed a higher virulence as determined by lethality and granuloma formation in mice than those with shorter ones. When purified glycolipid was injected into mice, granuloma formation and liver damage were most prominent with the glycolipid having longer carbon chain mycolic acid. Only a representative strain with longer carbon chain mycolic acid persisted in the spleen of mice after intravenous injection, while a strain with shorter carbon chain mycolic acid was readily eliminated. These results suggested that glycolipid was at least one of the virulence factors of R. equi and that the carbon chain length of mycolic acid might be critical in the expression of virulence.     

Crosslinked enzyme crystals of glucoamylase as a potent catalyst for biotransformations

Glucoamylase (E.C: 3.2.1.3, alpha-(1-->4)-glucan glucohydrolase) mainly hydrolyzes starch and has been extensively used in the starch, glucose (dextrose), and fermentation industries. Immobilized glucoamylase has an inherent disadvantage of lower conversion rates and low thermostability of less than 55 degrees C when used in continuous operations. We have developed crosslinked enzyme crystals (CLEC) of glucoamylase that overcome the above disadvantages, possess good thermal stability and retain 98.6% of their original activity at 70 degrees C for 1h, 77% activity at 80 degrees C for 1h, and 51.4% activity at 90 degrees C for 0.5h. CLEC glucoamylase has a specific activity of 0.0687 IU/mg and a yield of 50.7% of the original activity of the enzyme under optimum conditions with starch as the substrate. The crystals obtained are rhombohedral in shape having a size approximately 10-100 microm, a density of 1.8926 g/cm(3) and a surface area of 0.7867 m(2)/g. The pH optimum of the glucoamylase crystals was sharp at pH 4.5, unlike the soluble enzyme. The kinetic constants V(max) and K(m) exhibited a 10-fold increase as a consequence of crystallization and crosslinking. The continuous production of glucose from 10% soluble starch and 10% maltodextrin (12.5 DE) by a packed-bed reactor at 60 degrees C had a productivity of 110.58 g/L/h at a residence time of 7.6 min and 714.1g/L/h at a residence time of 3.4 min, respectively. The CLEC glucoamylase had a half-life of 10h with 4% starch substrate at 60 degrees C.     

Metallothionein induction as a potent means of radiation protection in mice

A striking resistance to lethal damage from a single dose of 6-8 Gy of X rays has been found in mice which had received various pretreatments to induce metallothionein (MT) synthesis in the liver prior to irradiation. Mice were injected with manganese (10 mg Mn/kg) or cadmium (3 mg Cd/kg) salt subcutaneously, or a patch of dorsal skin (2 X 2 cm2) was excised 1 or 2 days prior to irradiation. The increased tolerance of these mice to radiation was established by a marked decrease of mortality rate, an increase of mean survival time, a reduction of weight loss, and a smaller decrease in the number of leukocytes as compared with the control group. The LD50/30 for control mice was 6.3 Gy, while the corresponding values for the groups pretreated with Mn, Cd, and skin excision were 7.5, 7.7, and 7.9 Gy, respectively. The normal level of MT in mouse liver was approximately 25 micrograms/g tissue. This level increased 2.5- to 3-fold 24 h after 6.3 Gy irradiation. The MT levels of mice pretreated with Cd, Mn, and skin excision were increased 8-, 5-, and 7-fold, respectively, prior to irradiation as compared with the preirradiation control. These results indicate that the induction of MT in mouse liver is a significant factor in the mechanism of protection against radiation.     

Mapping genes encoding drug-metabolizing enzymes in recombinant inbred mice.

Probes for cytochrome P450IVA (P450IVA), alpha- and pi-class glutathione S-transferases (GST), and phenol-metabolizing UDP-glucuronyltransferase (UDPGT-K39) detected restriction fragment length variants (RFLVs) between C57BL/6J and DBA/2J mice. These variants were used to map the P450IVA genes (Cyp4 alpha) to chromosome 4, close to Mtv-13 and Pmv-19, midway between brown (b) and Gpd-1; GST alpha genes were mapped to chromosome 9, with a cross-hybridizing sequence mapping to another chromosome; the GST pi genes were mapped to the distal end of chromosome 1 near Pmv-21; one UDPGT-K39 variant to chromosome 1, between Acrg and Emv-17, and another showed linkage to Odc-10 on an unidentified chromosome. No RFLVs were detected with probes for P450IID, P450 reductase, androsterone-metabolizing UDPGT, GST mu, or microsomal GST.     

Use of 13C nuclear magnetic resonance spectroscopy for the evaluation of hepatic drug-metabolizing enzyme activity in perfused mouse livers

Experiments were performed to determine whether the activity of hepatic drug-metabolizing enzymes could be estimated in intact livers by 13C NMR spectroscopy. 13C-labelled aminopyrine was administered to isolated perfused mouse livers and the rate of decline in the NMR signal arising from the drug was used to estimate the half-life of aminopyrine. The technique was sufficiently sensitive to discriminate between the enzyme activities in normal livers and in livers whose enzyme activity was enhanced by treatment with sodium phenobarbitone. It was also demonstrated that demethylation of aminopyrine was not impaired in perfused livers from mice infected with Plasmodium berghei.     

Spatial order as a source of kinetic cooperativity in organized bound enzyme systems.

When enzyme molecules are distributed within a negatively charged matrix, the kinetics of the conversion of a negatively charged substrate into a product depends on the organization of fixed charges and bound enzyme molecules. Organization is taken to mean the existence of macroscopic heterogeneity in the distribution of fixed charge density, or of bound enzyme density, or of both. The degree of organization is quantitatively expressed by the monovariate moments of charge and enzyme distributions as well as by the bivariate moments of these two distributions. The overall reaction rate of the bound enzyme system may be expressed in terms of the monovariate moments of the charge density and of the bivariate moments of charge and enzyme densities. The monovariate moments of enzyme density do not affect the reaction rate. With respect to the situation where the fixed charges and enzyme molecules are randomly distributed in the matrix, the molecular organization, as expressed by these two types of moments, generates an increase or decrease of the overall reaction rate as well as a cooperativity of the kinetic response of the system. Thus both the alteration of the rate and the modulation of cooperativity are the consequence of a spatial organization of charges with respect to the enzyme molecules. The rate equations have been derived for different types of organization of fixed charges and enzyme molecules, namely, clustered charges and homogeneously distributed enzyme molecules, clustered enzyme molecules and homogeneously distributed charges, clusters of charges and clusters of enzymes that partly overlap, and clusters of enzymes and clusters of charges that are exactly superimposed. Computer simulations of these equations show how spatial molecular organization may modulate the overall reaction rate.     

Physicochemical characterization of the staphylolytic LysK enzyme in complexes with polycationic polymers as a potent antimicrobial

Staphylococcus aureus causes many serious visceral, skin, and respiratory diseases. About 90% of its clinical strains are multi-drug resistant, but the use of bacteriophage lytic enzymes offers a viable alternative to antibiotic therapy. LysK, the phage K endolysin, can lyse S. aureus when purified and exposed externally. It has been investigated in its complexes with polycationic polymers (poly-l-lysines (PLLs) of molecular weights 2.5, 9.6, and 55.2 kDa and their block copolymers with polyethylene glycol PLL₁₀–PEG₁₁₄, PLL₃₀–PEG₁₁₄, and PLL₃₀–PEG₂₃) as a basis for creating active and stable antimicrobial. Complexing with polycationic PLLs produces a stabilizing effect on LysK due to structure ordering in its molecules and break-down of aggregates as a result of electrostatic interaction. The stability of LysK in the presence of PLL–PEG block copolymers improves by both electrostatic and hydrophobic mechanisms. Complexes of LysK with 2.5, 9.6, 55.2 kDa poly-l-lysines and PLL₃₀–PEG₁₁₄ have demonstrated sufficient stability at the temperatures of physiological activity (37 °C) and storage (4 °C and 22 °C).     

Rational design of transition-state analogues as potent enzyme inhibitors with therapeutic applications.

The structures of the transition states for a variety of enzyme-catalyzed ribosyl group transfer reactions, determined by computational evaluation of multiple tritium and heavy atom kinetic isotope effects on these enzymatic reactions, have been found to show a considerable variation in the extent of bond cleavage at the ribosyl anomeric carbon. The calculated transition-state structures have been used to guide the design of high-affinity transition-state analogue inhibitors for 5'-methylthioadenosine nucleosidases with potential as therapeutic agents.