Basal cell carcinomas in association with basaloid follicular hamartoma.

We describe a unique case of various types of basal cell carcinoma (BCC) associated with basaloid follicular hamartoma (BFH) in a 56-year-old female patient. The lesion consisted of a dark brown and elastic soft nodule and papules within the area of a birthmark on the neck. The lesion was surgically excised. Histological examination of the nodular region revealed aggregations of neoplastic basaloid cells. We diagnosed the nodule as BCC with a racemiform or reticular pattern. In addition, a specimen taken from brownish black papules within the birthmark was found to be composed of anastomosing cords of basaloid cells accompanied by infundibular cystic structures. These features were consistent with an infundibulocystic BCC. In contrast, specimens from a hamartomatous plaque showed distinctive branching strands of basaloid cells that are suggestive of BFH. Therefore, our findings indicate that several types of BCC may develop within a BFH.

CD1a+, CD3+, CD4+, CD8+, CD68+ and cutaneous lymphocyte-associated antigen-positive cells in Bowen's disease.

BACKGROUND: Bowen's disease (BD) is a squamous cell carcinoma in situ that rarely invades into the underlying dermis. However, little is known about its immunohistology. Objectives To evaluate the relationship between the cytological properties of the tumour cells in BD and the host immune response. METHODS: We examined the expression of p53, proliferating cell nuclear antigen (PCNA) and Ki67 antigen, and the number of mitotic cells, together with the number of intratumoral and dermal infiltrating CD1a+, CD3+, CD4+, CD8+, CD68+ and cutaneous lymphocyte-associated antigen (CLA)+ cells in 18 cases of genital BD. RESULTS: When compared with normal genital skin (n = 10), there was a significantly higher number of mitotic cells as well as higher expression of p53+, PCNA+ and Ki67+ cells in BD. There was significant mutual correlation between CD3+, CD4+ and CD68+ cells in the tumoral epidermis. The number of CD1a+ Langerhans cells significantly decreased in BD epidermis; however, dermal CD1a+ cells were increased. Interestingly, numbers of dermal CD1a+ cells significantly correlated with those of intratumoral CD3+, CD4+ and CD68+ cells. In situ hybridization for human papillomavirus (HPV) demonstrated that HPV-infected BD had significantly less infiltration of intratumoral CD3+ cells and CLA+ cells. CONCLUSIONS: The present data suggest that dermal CD1a+ cells may participate in the immune surveillance and that HPV infection may interfere with the intratumoral infiltration of CLA+ cells in BD.

Novel metabolic pathway of estrone and 17beta-estradiol catalyzed by cytochrome P-450.

We have already reported that the quinol formation from some para-alkylphenols, which is a novel metabolic pathway catalyzed by cytochrome P-450, occurs in a rat liver microsomal system (). In the present study, we investigated whether estrone and 17beta-estadiol, each of which contains a p-alkylphenol moiety, are also oxidized into the corresponding quinols by cytochrome P-450. Six recombinant human cytochrome P-450 enzymes, CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2E1, and CYP3A4, were tested. The results show that estrone and 17beta-estadiol were converted into the corresponding quinols by CYP1A1, CYP2B6, and CYP2E1.

Antioxidant activity and xanthine oxidase inhibition activity of reductic acid: ascorbic acid analogue.

Reductic acid (2,3-dihydroxy-2-cyclopentenone, 1) decreased the ESR signal of 5,5-dimethyl-1-pyrroline 1-oxide (DMPO)-OH produced by hydroxyl radical and DMPO. 1 also inhibited lipid peroxidation initiated by cytochrome P450 and tert-butyl hydroperoxide. 1 inhibited xanthine oxidase activity, while ascorbic acid and 2-hydroxytetronic acid, an ascorbic acid analogue without side chain, did not.

Inhibition of E. coli growth by fullerene derivatives and inhibition mechanism.

Cationic ammonium fullerene derivatives (C60-bis(N, N-dimethylpyrrolidinium iodide) and C60-bis(N-methylpiperazinium iodide)) suppressed E. coli growth, whereas an anionic derivative (C60-dimalonic acid) did not. Both cationic derivatives inhibited E. coli dioxygen consumption. Inhibition of energy metabolism is concluded to be a mechanism of the growth inhibition effect of fullerene derivatives.

Inhibitory effect of a fullerene derivative, monomalonic acid C60, on nitric oxide-dependent relaxation of aortic smooth muscle.

1. Effects of a fullerene C60 derivative, monomalonic acid C60 (MMA C60), on endothelium-containing or denuded aorta of rabbit, trachea and ileum of guinea pig, and stomach (fundus), vas deferens and uterus of rat were studied pharmacologically. 2. MMA C60 (10(-5) M) significantly reduced the maximum response of the relaxation induced by acetylcholine in endothelium-containing thoracic aorta of rabbit, and the acetylcholine-induced relaxation was recovered in the presence of superoxide dismutase (SOD, 250 units/ml). 3. Nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, caused the relaxation of aorta without endothelium in a concentration-dependent manner, and the concentration-response curve was shifted to the right in the presence of MMA C60. This inhibitory effect of the derivative was also masked in the presence of superoxide dismutase (SOD). 4. Sodium nitroprusside-induced relaxation was not affected by either MMA C60 or SOD. In the other tissues, this C60 derivative had no effect on the responses induced by any agonist. 5. These observations indicate that MMA C60 inhibits the endothelium-dependent relaxation induced by acetylcholine but does not affect the agonist-induced contractile response of smooth muscle.

In vitro comparison between mouse B16 and human melanoma cell lines of the expression of ICAM-1 induced by cytokines and/or hyperthermia.

The expression of intercellular adhesion molecule-1 (ICAM-1) in mouse B16 and human melanoma cell lines was investigated by indirect immunofluorescence using a FACScan analyzer. Mouse B16 melanoma cell lines (B16-F1 and F10) did not express ICAM-1 under ordinary culture conditions. Neither in vitro hyperthermia at 41 degrees C for 3 or 6 hr nor cytokines such as gamma interferon (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) induced ICAM-1 expression in B16 melanoma cell lines. A combination of IFN-gamma with TNF-alpha caused slight induction of ICAM-1 expression in the B16-F10 melanoma cell line. Hyperthermia at 41 degrees C for 3 hr in combination with the cytokines induced a slight expression of ICAM-1 in the B16-F1 melanoma cell line. Hyperthermia at 41 degrees C for 3 hr or 6 hr did not induce de novo ICAM-1 expression but hyperthermia at 43 degrees C for 6 hr caused rather suppression of the expression of ICAM-1 in the three human melanoma cell lines tested. In contrast, they showed a clear increase in the expression of ICAM-1 after treatment with either with IFN-gamma or TNF-alpha, and the expression was further augmented by a combination of the two cytokines. Treatment with cytokines in combination with hyperthermia at 41 degrees C or 43 degrees C for 3 hr did not augment the expression of ICAM-1 over that in cytokine-treated human melanoma cell lines, at normal temperatures. Thus, it is concluded that mouse B16-F1 and F10 melanoma cell lines are different from human melanoma cell lines in terms of induction of ICAM-1 expression by cytokines and/or hyperthermia.

Kinetics of immunological parameters in patients with malignant melanoma treated with hyperthermic isolated limb perfusion.

Kinetics of immunological parameters such as natural killer (NK) cell activity and tritium-thymidine uptake rate of T lymphocytes by phytohemagglutinin stimulation were investigated using peripheral leukocyte fractions of melanoma patients treated with hyperthermic isolated limb perfusion (HILP). Also, serum concentrations of soluble intercellular adhesion molecule-1 (sICAM-1) were quantified during and after HILP. It was found that NK cell activity was augmented during HILP, and T lymphocyte function was stimulated 24 h and 1 week after HILP with statistical significance. NK cell activities in the cells isolated from perfused and non-perfused circulations were equally augmented during HILP in two patients examined. Serum concentrations of sICAM-1 in the patients who received HILP also increased 24 h or even 1 week after HILP. The stimulation of these immune competent cells and upregulation of sICAM-1 by HILP were independent of the stages of melanoma patients at the time of HILP or the doses of agents which were used for the infusion during HILP. The origin of cells which shed sICAM-1 into the serum of the patients who received HILP remains to be further investigated.

Inhibitory effects of a fullerene derivative, dimalonic acid C60, on nitric oxide-induced relaxation of rabbit aorta.

Dimalonic acid C60 (10(-5) M), a new fullerene derivative, produced an augmentation of phenylephrine-induced tone and reduced both the acetylcholine-induced maximum relaxation and the amplitude of substance P (10(-8) M)-induced relaxation in endothelium-containing thoracic aorta of rabbit; the acetylcholine- and substance P-induced relaxation was restored in the presence of superoxide dismutase (250 U/ml). Dimalonic acid C60 (10(-5) M) did not influence the phenylephrine-induced contractile response in the absence of endothelium, but the acetylcholine-induced relaxation was eliminated by removal of the endothelium. Superoxide anion generation, using hypoxanthine (1 mM)/xanthine oxidase (16 mU/ml), reduced the acetylcholine-induced relaxation and produced an augmentation of phenylephrine-induced tone in endothelium-containing strips; these effects were negated by the addition of superoxide dismutase (250 U/ml). A nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, caused relaxation of aorta without endothelium in a concentration-dependent manner, and the concentration-response curve was shifted to the right in the presence of dimalonic acid C60. This inhibitory effect of dimalonic acid C60 was also masked in the presence of superoxide dismutase. Sodium nitroprusside-induced relaxation was not affected by either dimalonic acid C60 or superoxide dismutase. These observations suggest that dimalonic acid C60 inhibits endothelium (nitric oxide)-dependent agonist-induced relaxation through the production of superoxide.

Substituent elimination from p-substituted phenols by cytochrome P450. ipso-Substitution by the oxygen atom of the active species.

When various p-substituted phenols (substituent = NO2, CN, CH2OH, COCH3, COPh, COOH, F, Cl, and Br) were incubated with rat liver microsomes, the substituent was eliminated to produce hydroquinone, and the reaction was inhibited by CO and a cytochrome P450-specific inhibitor. In the case of p-cresol (substituent = CH3), p-toluquinol was formed instead of hydroquinone. Experiments using 18O2 proved that the elimination is accompanied with ipso-substitution by the oxygen atom of the active species in cytochrome P450. These results are similar to those in a cytochrome P450. These results are similar to those in a cytochrome P450 chemical model system (Ohe, T., et al., Tetrahedron Lett. 42, 7681-7684, 1995), implying that the model is a good mimic of cytochrome P450. Substrates that lack a hydroxy group, namely p-substituted toluenes, did not undergo the reaction, thus indicating that a hydroxy group at the p-position to the eliminated substituent is necessary for this pathway. This is the same as the result obtained with the cytochrome P450 model. Finally, to elucidate how the substituent is eliminated, we attempted to detect the product derived from the eliminated group with several substrates. Results indicated that the mechanism of the substituent elimination can be divided into two types: the substituent is eliminated as an anion or as a cation.

Novel metabolic pathway of arylethers by cytochrome P450: cleavage of the oxygen-aromatic ring bond accompanying ipso-substitution by the oxygen atom of the active species in cytochrome P450 models and cytochrome P450.

We have found a novel metabolic pathway of arylethers, involving the cleavage of the oxygen-aromatic ring bond. When p-(p-nitrophenoxy)phenol was utilized as a substrate, cleaved products, p-nitrophenol and p-benzoquinone, were formed in two cytochrome P450 model systems, meso-tetraphenylporphinatoiron(III) chloride-NaBH4/O2 system and meso-tetrakis (2,6-difluorophenyl)porphinatoiron(III) chloride-m-chloroperoxybenzoic acid (mCPBA) system. Rat liver microsomes also catalyzed this reaction, which was inhibited by a cytochrome P450-specific inhibitor, and it was confirmed that this cleavage proceeded in vivo. Further, experiments using [18O]mCPBA and 18O2 proved that the cleavage reaction is accompanied with the ipso-substitution by the oxygen atom of the active species in both cytochrome P450 model system and cytochrome P450. When the microsomal reactions of p-(p-nitrophenoxy)phenol analogues which lack a hydroxy group, namely p-nitrophenoxybenzene, p-(p-nitrophenoxy)anisole, and p-(p-nitrophenoxy)toluene, were investigated, the cleavage reaction occurred via p-(p-nitrophenoxy)phenol in the cases of p-nitrophenoxybenzene and p-(p-nitrophenoxy)anisole, indicating that a hydroxy group at the p-position to the ether bond is necessary for this pathway. This metabolic pathway appears to be important, because a diarylether linkage, which is very stable and has generally been thought to resist metabolism, is cleaved and benzoquinone, a highly toxic metabolite, is formed.

Application of chemical cytochrome P-450 model systems to studies on drug metabolism. VI. N,N-coupling reaction of N-methylaniline catalyzed by polypeptide-bound porphyrinatoiron(III) and cytochrome P-450.

Polypeptide-bound porphyrinatoiron(III) effectively catalyzed the N,N-coupling reaction of N-methylaniline in the presence of NaBH4/O2/tetramethyl-ammonium hydroxide or cumenehydroperoxide, whereas the corresponding non-bound monomer porphyrinatoiron(III) had little catalytic activity. Rat liver microsomes also catalyzed the N,N-coupling which was inhibited by cytochrome P-450 specific inhibitors, SKF-525A and metyrapone. The polypeptide-bound porphinatoiron(III) appears to be an excellent cytochrome P-450 model for drug metabolism studies.

Cytochrome P450-like substrate oxidation catalyzed by cytochrome c and immobilized cytochrome c.

Cytochrome c (cyt.c) was shown to catalyze cytochrome P450 (P450)-like oxidative reactions, such as N-, and O-demethylation, S-oxidation, and epoxidation of olefins. A more detailed examination showed that (i) N-methylcarbazole and thioanisole oxidation with H2(18)O2 catalyzed by cyt.c resulted in introduction of 18O into the product, and (ii) during the epoxidation of cis-stilbene catalyzed by cyt.c, the stereochemistry of the substrate was retained and 18O was introduced when H2(18)O2 was used as an oxidant. These results show that cyt.c catalyzed N-demethylation, S-oxidation, and epoxidation in the same manner as P450. To utilize these P450-like reactivities effectively, cyt.c was immobilized on poly-gamma-methyl-L-glutamate. Up to 99% of the cyt.c used was immobilized. This immobilized cyt.c catalyzed N-demethylation, S-oxidation, and epoxidation in the same manner as both P450 and free cyt.c, and the activities of these reactions were increased by the immobilization. In N-demethylation of N,N-dimethylaniline with cumene hydroperoxide (CHP) catalyzed by cyt.c, the Vmax for CHP was increased by 4.4-fold by the immobilization of the enzyme, while the Km remained unchanged. Since P450 is involved in the metabolism of many xenobiotics, the above results suggest that immobilized cyt.c may be useful in drug metabolism research.

Metabolism and penetration through blood-brain barrier of parkinsonism-related compounds. 1,2,3,4-Tetrahydroisoquinoline and 1-methyl-1,2,3,4-tetrahydroisoquinoline.

14C-Labeled 1,2,3,4-tetrahydroisoquinoline (TIQ) and 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) were synthesized, and their metabolism and tissue distribution were studied. Both compounds showed similar metabolic patterns. In 24 hr after po administration (50 mg/kg) to rats, 76% of TIQ and 72% of 1MeTIQ were excreted unchanged, and 2.7 and 8.7% were excreted as the 4-hydroxyl derivatives, 4-hydroxy-TIQ and 4-hydroxy-1MeTIQ, respectively. Small amounts of N-methylated metabolites, 2-methyl-TIQ (0.4%) and 2-methyl-1MeTIQ (0.7%) were detected. Isoquinoline (2.5%) also was found as a metabolite of TIQ and 1-methyl-3,4-dihydroisoquinoline (1.0%) was found as a metabolite of 1MeTIQ. The concentration of labeled compounds in the brain was about 4.5-fold higher than the blood concentration at 4 hr after dosing, and over 90% was unchanged TIQ or 1MeTIQ. These data indicated that TIQ and 1MeTIQ easily passed through the blood-brain barrier and were concentrated in the brain. Thus, it appears that TIQ and 1MeTIQ as endogenous or exogenous amines may accumulate in the brain and may be related to the onset of Parkinson's disease.

NADPH mediates the inactivation of bovine liver catalase by monochloroamine.

Bovine liver catalase suffered a biphasic inactivation when exposed to NH2Cl. A rapid and irreversible phase of activity loss was followed by a much slower and reversible inactivation. Removal of tightly bound NADPH from the enzyme decreased the extent of the rapid phase; whereas addition of NADPH augmented it. The catalase from Aspergillus niger, which does not contain bound NADPH, was not nearly as sensitive toward NH2Cl as was the liver enzyme and was not sensitized by added NADPH. NADPH is oxidized by NH2Cl, as evidenced by loss of the 340-nm absorption band, but the product of this oxidation was not NADP+. The rapid inactivation of liver catalase by NH2Cl was accompanied by some bleaching of the bands in the visible, while the slow inactivation was coincident with the appearance of a new band at 570 nm. A tentative explanation for these observations is proposed.

Reactions of hypochlorite with catalase.

OCl-/HOCl imposed a rapid inactivation of catalase (hydrogen-peroxide: hydrogen-peroxide oxidoreductase, EC 1.11.1.6), some of which was slowly reversible upon subsequent exposure to H2O2. Ethanol accelerated this restoration of activity by H2O2. OCl- caused biphasic changes in the visible absorption spectrum of catalase, which were partially reversed by dithionite. A scheme of reactions involving axial ligation of one or two OCl- to heme iron, followed by heterolytic or homolytic cleavages of the O-Cl bond, is proposed to account for the behavior of the system.

Effects of urea and trimethylamine-N-oxide on enzyme activity and stability.

The interactions of urea, trimethylamine-N-oxide (TMAO), and related solutes on a number of enzymes were examined. Urea inhibited enzymatic activity and accelerated the thermal inactivation of catalase, whereas TMAO activated some enzymes but inhibited others. The effects of urea and of TMAO, whether parallel or in opposition, were exerted independently. Thus, in those cases where TMAO increases enzymatic activity, it did so to the same relative degree, whether or not urea was present. TMAO markedly decreased the rate of thermal inactivation of catalase, indicating that it does favor compact protein structures. The assumption that TMAO factors compaction of protein structure, whereas urea has the contrary effect, does not lead to the expectation that TMAO must always oppose the effect of urea on enzymatic activity, since the most compact form of an enzyme may not always be the most active form.

An activity stain for dihydroxy-acid dehydratase.

An activity stain has been devised for the dihydroxy-acid dehydratase. When applied to polyacrylamide gel electropherograms of crude soluble extracts of Escherichia coli, it detected a single electromorph. The intensity of staining increased with the amount of extract protein applied to the gel. Activity staining demonstrated that (a) anaerobically grown cells contain more extractable dehydratase activity than do aerobically grown cells; (b) exposure of E. coli to 4.2 atm O2 caused virtually complete loss of activity; (c) exposure of cells to paraquat or plumbagin in the presence of dioxygen, but not in its absence, caused a massive loss of activity. These data illustrate the utility of this activity stain and demonstrate that the dehydratase is inactivated by O2- generated within cells.

Superoxide radical initiates the autoxidation of dihydroxyacetone.

The aerobic xanthine oxidase reaction causes the cooxidation of dihydroxyacetone in a process which is strongly inhibited by superoxide dismutase but not by catalase, HO X scavengers, or iron-inactivating chelating agents. Several molecules of the sugar can be oxidized per O2- introduced. A free radical chain mechanism, in which O2- acts both as an initiator and as a chain propagator, is proposed. Simple sugars capable of tautomerizing to enediols may now be added to the list of biologically relevant targets for O2-.