Spindle cell carcinoma arising in the pharynx, with granulocytosis and high serum granulocyte colony stimulating factor titre.

We report the case of a 67-year-old man with a left pharyngeal tumour, whose peripheral blood showed granulocytosis (white blood cell count, 58,300/microl) and a high serum granulocyte colony stimulating factor titre (184 pg/ml). The tumour showed pleomorphic proliferation of atypical spindle cells in a myxomatous stroma, revealing a sarcomatous pattern. The spindle-shaped neoplastic cells had irregularly shaped nuclei, a thick nuclear membrane, prominent eosinophilic nucleoli and abundant cytoplasm. They strongly expressed wide-spectrum keratin, cytokeratins (CAM5.2, MNF116), vimentin and vascular endothelial growth factor. A few neoplastic cells expressed granulocyte colony stimulating factor. A spindle cell carcinoma was diagnosed. This may be the first documented case of a granulocyte colony stimulating factor producing cancer arising in the pharynx. The patient died four months after the initial symptoms appeared.

Cycloaddition of 2(1H)-pyridones having a methoxycarbonyl group to 1,3-butadiene derivatives.

The cycloaddition of 4-methoxycarbonyl-2(1H)-pyridones to silyloxydienes gave isoquinolone derivatives in reasonable yields. Furthermore, the cycloaddition of 6-methoxycarbonyl-2(1H)-pyridones to 2,3-dimethyl-1,3-butadiene produced cycloadducts (isoquinolone and quinolone derivatives) and double cycloadducts (phenanthridone derivatives). The activation energies using Gaussian 98 with RHF/3-21G level of 4- and 6-methoxycarbonyl-2(1H)-pyridones coincided with the experimental facts.

Synthesis of isoquinoline derivatives by Diels-Alder reactions of 2(1H)-pyridones having an electron-withdrawing group with methoxy-1,3-butadienes.

Diels-Alder (DA) reactions of 2(1H)-pyridones having an electron-withdrawing group at the 4-position with 2,3-dimethoxy- and 2-methoxy-1,3-butadienes gave isoquinoline derivatives. Furthermore, an isoquinoline alkaloid (6,7-dimethoxy-2-methyl-1(2H)-isoquinolone) was synthesized by elimination of hydrogen cyanide and dehydrogenation of the DA-adduct having a cyano group at the 4a-position.

[Diabetic changes in the stria vascularis in humans--a study of PAS-stained temporal bone sections].

Sensorineural hearing loss can be caused by diabetes mellitus, and diabetic microangiopathy contributes to diabetic complications such as nephropathy. I compared the outer diameter of strial capillaries, the strial atrophy rate, and basement membrane thickening in the strial capillaries of temporal bone sections from 16 diabetics and 16 non-diabetics matched for age and sex and assessed the correlations between these values and age, duration of disease, fasting blood sugar, and glycohemoglobin. In the non-diabetic group, the minimum and maximum outer diameters of the capillary in the cochlear apical turn were larger than in the basal and middle turns. The strial atrophy rate in the apical turn was higher than in the basal and middle turns. There was no significant difference in basement membrane width between the turns. In the diabetic group, there were no significant differences in minimum outer diameter and basement membrane width between the turns. The maximum outer diameter of the middle turn was larger than that of basal turn. The strial atrophy rate in the apical turn was higher than in the basal and middle turn. In the comparison between the diabetic group and the non-diabetic group, the maximum outer diameter of the apical turn in the diabetics was smaller than in the non-diabetics, the basement membrane width in the basal, apical and all three turns as a whole in the diabetics was thicker than in the non-diabetics, the strial atrophy rate in the basal turn and all three turns as a whole in diabetics was higher than in non-diabetics. There were two correlations in the non-diabetics, between age and strial atrophy rate and between age and basement membrane width in the basal turn, and there were positive correlations in the diabetics, between the strial atrophy rate and fasting blood sugar and between strial atrophy rate and glycohemoglobin in the basal turn. Because of basement membrane thickening toward capillary lumen, these results led to the hypothesis that strial atrophy is one factor in diabetic hearing loss.

Development of microplate-radioluminography for 32P.

Basic research for designing a microplate which can be applied to determination of 32P by radioluminography was carried out. It was proven that 32P can be determined by using the microplate which has plural planchets placed at intervals on a thin plate and a brass collimator that fills the gaps between the planchets. The microplate is joined with the collimator, and exposed to an imaging plate for 24 h. The detection limit of the proposed method was estimated to be 6 m Bq. The cross-talk ratio was negligibly small (0.2%).

Suppression and enhancement of dye lasing and stimulated Raman scattering from various dye-doped liquid spheres.

The observation of suppression or enhancement of dye lasing and stimulated Raman scattering (SRS) from various dye-doped liquid droplets, in which SRS from the initial pumping wavelength appeared in shorter and longer wavelengths of various dye fluorescence regions, is reported; SRS from the dye-lasing wavelengths (double resonances) and stimulated resonance Raman scattering of dyes are included. Furthermore, the contribution to SRS of the dye fluorescence (depending on dye concentration and different dyes) and dye-lasing suppression that is due to stimulated resonance Raman scattering is also described.

A novel pathway for formation of thiol metabolites and cysteine conjugates from cysteine conjugate sulphoxides.

p-Bromothiophenol and S-(p-bromophenyl)-L-cysteine were formed enzymatically from S-(p-bromophenyl)-L-cysteine sulphoxide in the in vitro systems with isolated rat hepatocytes or purified cysteine conjugate beta-lyases. Isotope dilution study with non-radiolabelled carrier of each product suggested the initial liberation of the thiol and subsequent formation of the cysteine conjugate. C-S bond cleavage pathway to liberate sulphenic acid and thiol are postulated to play an important role in in vivo generation of toxic intermediates and products from cysteine conjugates.

Purification and characterization of 3-mercaptolactic acid S-conjugate oxidases.

Two enzymes catalysing the oxidative formation of 3-mercaptopyruvic acid S-conjugates from L-3-mercaptolactic acid S-conjugates were purified to apparent homogeneity from rat liver cytosol. The two enzymes, tentatively designated MLO-I and MLO-II, showed a molecular mass of 160 and 250 kDa and were composed of four and six subunits of 41 and 39 kDa, respectively. Both enzymes possessed flavin mononucleotide as prosthetic group and oxidized several aromatic and aliphatic S-substituted L-3-mercaptolactic acids as well as alpha-hydroxy acids such as L-3-phenyllactic acid and L-2-hydroxyisocaproic acid. Glycolic acid and 3-(4-hydroxyphenyl)-lactic acid were the specific substrates for MLO-I and MLO-II, respectively. Neither of the enzymes oxidized beta- and gamma-hydroxy acids such as 3- and 4-hydroxybutyric acid. 2-Hydroxyisobutyric acid, ethyl-2-hydroxybutyrate, malic acid, 1-butanol, benzyl alcohol and L-leucine did not act as substrates for the enzymes. MLO-I and MLO-II exerted their maximum activities around pH 5.5 with Km of 0.5 and 0.25 mM and Vmax of 0.9 and 0.2 mumol/min/mg, respectively, when S-(4-bromophenyl)-3-thiolactic acid was used as substrate. MLO-I was inhibited by sulphydryl-modifying agents, while MLO-II was not. Both enzymes were strongly inhibited by divalent metal ions. These results indicate that MLO-I and MLO-II are different from L-amino acid oxidase (EC 1.4.3.2), malate oxidase (EC 1.1.3.3), L-alpha-hydroxy acid oxidase (EC 1.1.3.15) and glycolate oxidase (EC 1.1.3.1). The present enzymes are likely to be involved in the formation of cysteine conjugates from L-3-mercaptolactic acid S-conjugates in conjunction with cysteine conjugate aminotransferases.

Specific induction by glucocorticoids of steroid esterase in rat hepatic microsomes and its release into serum.

Steroid esterase hydrolysing methylprednisolone 21-hemisuccinate was induced specifically and markedly in hepatic microsomes and serum of rats by various glucocorticoids. Among the glucocorticoids examined, dexamethasone and betamethasone showed the highest potency to induce the hepatic steroid esterase, the induction ratio being 32 and 33 times higher than the basal level (about 160 mU/g liver), respectively. Steroid esterase in the serum was induced greatly by fluocinolone acetonide and betamethasone to 92 and 79 times of the basal level of about 16 mU/mL, respectively, followed by dexamethasone and methylprednisolone. When dexamethasone was given to rats, the enzyme in other tissues except for duodenum and small intestine (of which activity was lowered to 50% of the basal level) was also elevated, but the induction ratio was much lower than that in the liver and serum. The induction of the steroid esterase is probably due to stimulation of de novo synthesis of the enzyme by glucocorticoids, because the elevation of esterase activity was inhibited by treatment with cycloheximide (a translation inhibitor) and actinomycin D (a transcription inhibitor), and about 4- and 10-hr lag time was observed before the elevation of esterase activity in liver and serum, respectively. Coupled with these observations the following results indicate that the steroid esterase in serum is probably synthesized in the liver and subsequently released into the blood via the Golgi apparatus: (1) when the liver of rats treated with dexamethasone was subjected to perfusion with a recycling system, significant amounts of steroid esterase were released into the perfusate; (2) anti-hepatic esterase antibody inhibited the steroid esterase activity not only in the liver but also in serum; and (3) monensin, which prevents the secretion of various kinds of secretory proteins by disrupting the function of the Golgi apparatus, inhibited the elevation of the steroid esterase activity in serum by dexamethasone but did not affect the induction in liver.

Purification and characterization of glucocorticoid-inducible steroid esterase in rat hepatic microsomes.

A steroid esterase hydrolysing methylprednisolone 21-hemisuccinate was purified from the hepatic microsomes of rats treated with dexamethasone, a potent inducer of the esterase. The enzyme was solubilized by Lubrol WX and purified up to 30-fold over the microsomal fraction by ammonium sulfate fractionation and successive chromatographies with gel permeation, DEAE-cellulose and hydroxylapatite. The steroid esterase thus purified showed a single band and a molecular mass of 58 kDa on SDS-polyacrylamide gel electrophoregram. The enzyme appears likely to exist as two interconvertible forms, which can be distinguished by pI values, pI 4.9 and 5.1. The enzyme was completely inhibited by organic phosphates, indicating that it can be classed as a carboxylesterase (EC 3.1.1.1). Both negatively charged and uncharged esters of several steroids (methylprednisolone, hydrocortisone, deoxycorticosterone and dehydrotestosterone) as well as various non-steroidal esters including 4-nitrophenyl esters were hydrolysed by the enzyme, but none of the amides were substrates. The enzyme showed higher activity with increasing lipophilicity of the substrates. It is noticeable that the optimum pH for charged esters was 5.5, whereas the highest activity was observed around pH 7-8 for uncharged esters. When methylprednisolone 21-hemisuccinate (one of the charged esters) was used as substrate, the Km value was 2.8 mM and Vmax was 59.3 mumol/mg protein for 1 min at the optimum pH of 5.5. Regarding the methyl ester of methylprednisolone 21-hemisuccinate, Km and Vmax values were 1.8 mM and 193 mumol/mg protein/min, respectively, at the optimum pH of 7.0. On the basis of these results, the enzyme is most likely a carboxylesterase.

Occurrence of cholesterol 7 alpha- and 7 beta-hydroperoxides in rat skin as aging markers.

Evidence for presence of cholesterol 7 alpha- and 7 beta-hydroperoxides in rat skin was presented for the first time. The 7-hydroperoxides in rat skin were reduced with sodium borohydride and trimethylsilylated for identification with the authentic compounds by gas chromatography/mass spectrometry. A content of cholesterol 7-hydroperoxides in rat skin, determined by high performance liquid chromatography with a chemiluminescence detector, highly correlated with the age of rats (r = 0.874; between 1 and 45 weeks old), indicating that cholesterol 7 alpha- and 7 beta-hydroperoxides were good markers for aging.

Purification and characterization of 3-mercaptopyruvic acid S-conjugate reductases.

Three kinds of 3-mercaptopyruvic acid S-conjugate reductase (MPR-I, MPR-II and MPR-III) were purified from rat liver cytosol. These enzymes reduced 3-mercaptopyruvic acid S-conjugates derived from cysteine conjugates and some endogenous alpha-keto acids to the corresponding alpha-hydroxy acids in the presence of either NADH (for MPR-I and MPR-II) or NADPH (MPR-III), while simple aldehydes or ketones did not significantly induce substrate activity. The molecular weight of the present enzymes was about 80 kDa composed of two subunits of the same molecular weight. Km values of MPR-I, MPR-II and MPR-III were 0.38, 0.06 and 0.29 mM for S-(4-bromophenyl)-3-thiopyruvic acid, respectively, and 0.15 mM for NADH (MPR-I, MPR-II) and NADPH (MPR-III). Vmax values of MPR-I, MPR-II and MPR-III for this substrate were 5.3, 20 and 13 nmol/min/mg, respectively. The sulphydryl-modifying agents inhibited the enzyme activities of all the three reductases. Based on the properties including substrate selectivity for alpha-keto acids derived from aromatic amino acids, we assumed that MPR-II and aromatic alpha-keto acid reductase are the same enzyme, while enzymes similar to MPR-I and MPR-III have not been reported. From the viewpoints of metabolism of xenobiotics, these enzymes are likely to be important in biotransformation of cysteine conjugates to 3-mercaptolactic acid S-conjugates.

Oxidation of tenoxicam by leukocyte peroxidases and H2O2 produces novel products.

A leukocyte extract, which had a high peroxidase activity (mostly myeloperoxidase), converted tenoxicam [4-hydroxy-N-(2'-pyridyl)-2-methyl-2H-thieno-(2,3e)-1,2-thiazine-3 - carboxamide-1,1-dioxide] a potent antiinflammatory drug, into four novel metabolites in the presence of H2O2: 4,5-dihydro-4-oxo-5-methyliminopyrido (1,2a) imidazole (metabolite I), 2-carboxyl-3-thiofenesulfinic acid (metabolite II), 2-carboxyl-3-thiofenesulfonic acid (metabolite III), and N-methyl-N'-(2-pyridyl)oxamide (metabolite IV). These metabolites were probably formed by a one-electron oxidation reaction at the center carbon atom of the beta-diketone moiety of tenoxicam. Tenoxicam is a cofactor for the reduction of peroxidases and this capability may explain at least a part of the antiinflammatory effect of tenoxicam.

Purification and characterization of cysteine conjugate transaminases from rat liver.

1. Soluble cysteine-conjugate alpha-ketoglutarate transaminase (CAT-I) was purified about 670-fold from rat liver cytosol using s-(p-bromophenyl)-L-cysteine as amino acid substrate. The enzyme preparation of the final step of purification showed a single band in polyacrylamide gel electrophoresis. CAT-I accounted for 64% of the transaminase activity in cytosol. 2. The mol. wt of the enzyme was about 64,000 as determined by gel filtration. Respective Km values for s-(p-bromophenyl)-L-cysteine and alpha-ketoglutaric acid were 1.0 and 1.3 mM in Tris-acetate buffer (pH 7.0). Aminooxyacetic acid, hydroxylamine, and KCN inhibited the enzyme activity. 3. In addition to CAT-I, two isozymes (CAT-IIA and CAT-IIB) were partially purified from rat liver cytosol. In respect of mol. wt, substrate specificity towards cysteine conjugates, and several other properties, CAT-IIA and CAT-IIB were very similar to CAT-I. However, differences were observed for these enzymes in the rate of reverse reaction (formation reaction of cysteine conjugates and alpha-ketoglutaric acid) and substrate specificity towards L-aspartic acid and L-cysteinesulphinic acid.

Involvement of cystathionase in the formation of alkane-thiols from corresponding cysteine conjugates.

1. Rat liver cystathionase [EC.4.4.1.1] mediated a cleavage reaction of the C-S bond of S-alkyl-L-cysteine conjugates to give equimolar amounts of corresponding thiols, ammonia, and pyruvic acid. Neither S-aryl- nor S-aralkyl-L-cysteine conjugates were acceptable substrates. 2. The Km value for S-(tert-butyl)-L-cysteine was 0.3 mM at pH 8.5 in Tris-HCl buffer. 3. The S-alkyl-L-cysteine lyase activity of cystathionase was inhibited with carbonyl reagents and dithiothreitol. 4. The present finding that cystathionase has activity towards cysteine conjugates may provide some insights into the role of this enzyme in the metabolism of xenobiotics.

Transamination of LTE4 by cysteine conjugate aminotransferase.

Leukotriene E4 (LTE4) was transaminated to 5(S)-hydroxy-6(R)-S-(2-keto-3-thiopropionyl)-7,9-trans-11,14-cis- eicosatetraenoic acid (tentatively designated as LTG4) by cysteine conjugate aminotransferase I purified from rat liver supernatant in the presence of alpha-ketoglutaric acid or alpha-keto-gamma-methiolbutyric acid. The transamination activity was present in the kidney as well as in the liver, but not in the lung or leukocytes.

Purification and properties of methyl sulfoxide reductases from rat kidney.

Two kinds of enzymes (tentatively designated methyl sulfoxide reductases I and II) responsible for the reduction of the methyl sulfoxide group on various xenobiotics have been purified about 223- and 155-fold, respectively, from rat kidney cytosol. The molecular weight was determined to be 12,000 +/- 1000 for methyl sulfoxide reductase I and 24,000 +/- 1000 for methyl sulfoxide reductase II. Thioredoxin or dithiothreitol is essential in order for the reducing activity to occur. The respective Km values of p-bromophenylmethyl sulfoxide were 2.75 and 1.30 mM for methyl sulfoxide reductases I and II. Replacement of the methyl group on the sulfur atom with a longer alkyl group or phenyl group caused a markedly low or negligible substrate activity.