Target organ-specific inactivation of drug metabolizing enzymes in kidney of hamsters treated with estradiol

Chronic treatment of hamsters with estradiol for several months has previously been shown to decrease the specific content of cytochrome P450 in the kidney, a target of hormonal carcinogenesis, but not in liver. The reason for this decrease in metabolic enzyme activity is unknown and has been examined in this investigation. We now report that the decrease in specific content of renal cytochrome P450 by 73% in response to estradiol was not affected by co-treatment with tamoxifen for 1 month. The subcutaneous infusion of 250 μg/day estradiol for 7 days lowered renal cytochrome P450 by 71% from control values and was therefore used for further mechanistic studies. This treatment decreased renal activities of estradiol 2- or 4-hydroxylase by 77 to 80%, of 7-ethoxycoumarin-O-deethylase by 66% of control values, respectively, and completely eliminated aryl hydrocarbon hydroxylase activities, whereas liver enzymes remained unaffected. After 7 days of infusion of estradiol, fluorescent products of lipid peroxidation were more than doubled in hamster kidney but remained unchanged in liver. The possibility of enzyme destruction by binding of estradiol 2,3-quinone to metabolizing enzymes was investigatedin vitro. In the presence of 2-hydroxyestradiol, cumene hydroperoxide, and microsomes, conditions known to favor the oxidation of the steroid to quinone, the binding of catechol estrogen metabolite to microsomal protein increased 60 fold over control values in the absence of cofactor. Purified rat liver cytochrome P450c also oxidized 2-hydroxyestradiol to 2,3-estradiol quinone. The rate of oxidation was linear for the first 2–3 min, but thereafter decreased with time. Under these incubation conditions, irreversible binding of catechol estrogen metabolite to cytochrome P450c increased for the first 2–3 min and then remained at this plateau level. It was concluded that enzyme destruction by a reactive estrogen metabolite or by lipid peroxides may be a major reason for the organ-specific decrease in cytochrome P450 enzymes in kidneys of estrogen-treated hamsters.     

Clostridium neopropionicum sp. nov., a strict anaerobic bacterium fermenting ethanol to propionate through acrylate pathway

Strain X4 was isolated several years ago from an anaerobic mesophilic plant treating vegetable cannery waste waters. It was the first example of propionic fermentation from ethanol. Morphologic and physiologic characterizations of the strain are presented here. This strain is described as type strain of a new species, Clostridium neopropionicum sp. nov. Whole cells of strain X4 ferment [1-¹³C]ethanol and CO₂ to [2-¹³C]propionate, [1-¹³C]acetate and [2-¹³C]propanol, suggesting the absence of a randomizing pathway during the propionate formation. Enzymes involved in this fermentation were assayed in cell-free extracts of cells grown with ethanol as sole substrate. Alcohol dehydrogenase, aldehyde dehydrogenase, phosphate acetyl transferase, acetate kinase, pyruvate synthase, lactate dehydrogenases, and the enzymes of the acrylate pathway were detected at activities sufficient to be involved in ethanol fermentation. The same pathway may be used for the degradation of lactate or acrylate to acetate.     

Regulation of ACV synthetase activity in the beta-lactam biosynthetic pathway by carbon sources and their metabolites

The activity of -l--aminoadipyl)-l-cysteinyl-d-valine catalysis. Addition of l-cysteine to fermentation media increased -lactam production in both organisms and alleviated the negative carbon source regulation by glycerol in S. clavuligerus.     

Anaerobic degradation of trans-cinnamate and ω-phenylalkane carboxylic acids by the photosynthetic bacterium Rhodopseudomonas palustris: evidence for a β-oxidation mechanism

The mechanism responsible for the initial steps in the anaerobic degradation of trans-cinnamate and -phenylalkane carboxylates by the purple non-sulphur photosynthetic bacterium Rhodopseudomonas palustris was investigated. Phenylacetate did not support growth and there was a marked CO₂ dependence for growth on acids with greater side-chain lengths. Here, CO₂ was presumably acting as a redox sink for the disposal of excess reducing equivalents. Growth on benzoate did not require the addition of exogenous CO₂. Aromatic acids with an odd number of side-chain carbon atoms (3-phenylpropionate, 5-phenylvalerate, 7-phenylheptanoate) gave greater apparent molar growth yields than those with an even number of side-chain carbon atoms (4-phenylbutyrate, 6-phenylhexanoate, 8-phenyloctanoate). HPLC analysis revealed that phenylacetate accumulated and persisted in the culture medium during growth on these latter compounds. Cinnamate and benzoate transiently accumulated in the culture medium during growth on 3-phenylpropionate, and benzoate alone accumulated transiently during the course of trans-cinnamate degradation. The transient accumulation of 4-phenyl-2-butenoic acid occurred during growth on 4-phenylbutyrate, and phenylacetate accumulated to a 1:1 molar stoichiometry with the initial 4-phenylbutyrate concentration. It is proposed that the initial steps in the anaerobic degradation of trans-cinnamate and the group of acids from 3-phenylpropionate to 8-phenyloctanoate involves -oxidation of the side-chain.Abbreviation 3-PP 3-phenylpropionic acid - 4-PB 4-phenylbutyric acid - 5-PV 5-phenylvaleric acid - 6-PH 6-phenylhexanoic acid - 7-PH 7-phenylheptanoic acid - 8-PO 8-phenyloctanoic acid - 4-P2B 4-phenyl-2-butenoic acid - GC/MS Gas chromatography/Mass spectrometry - HPLC High-pressure liquid chromatography     

Degradation of 4-nitrobenzoate via 4-hydroxylaminobenzoate and 3,4-dihydroxybenzoate in Comamonas acidovorans NBA-10

Comamonas acidovorans NBA-10 was previously shown to degrade 4-nitrobenzoate via 4-hydroxylaminobenzoate and 3,4-dihydroxybenzoate. Washed cells, grown on a mixture of 4-nitrobenzoate and ethanol, stoichiometrically produced ammonium and 3,4-dihydroxybenzoate from 4-nitrobenzoate under anaerobic conditions provided ethanol was present. In cell extracts 4-hydroxylaminobenzoate was degraded to ammonium and 3,4-dihydroxybenzoate, but this activity was lost upon dialysis. No requirement for a cofactor was found, but rather reduced incubation conditions were necessary to restore enzyme activity. The 4-hydroxylamino-degrading enzyme was purified and the role of this novel type of enzyme in the degradation of nitroaromatic compounds is discussed.Abbreviation 4-ABA 4-aminobenzoate - 4-NBA 4-nitrobenzoate - 4-HABA 4-hydroxylaminobenzoate - 3,4-diHBA 3,4-dihydroxybenzoate     

In search of specific cytotoxic T lymphocytes infiltrating or accompanying human ovarian carcinoma

Summary Lymphocytes infiltrating human ovarian carcinoma obtained directly from the tumour mass (tumour-infiltrating lymphocytes, TIL) or from the carcinomatous ascites (tumour-associated lymphocytes, TAL) were expanded in vitro in long-term cultures with interleukin-2 and tested for their specific cytolytic activity. Killing of the autologous tumour was detected only in a proportion of the patients, less frequently in TIL compared to TAL. In fact two out of ten TIL and four out of nine TAL cultures tested showed significant levels of lysis against the autologous tumour. This cytotoxic activity was not restricted to the autologous tumour, as other tumour cell lines, including non-ovarian ones, were lysed as well. The cultures that were not cytotoxic against the autologous tumour were in most cases able to lyse other tumour cell lines of ovarian or other histology. Cloning of TIL from one patient was performed: of 22 clones tested, 4 displayed higher cytotoxicity against the autologous tumour compared to the uncloned population and 3 out of these 4 did not kill an irrelevant carcinoma cell line. In order to stimulate the expansion of putative specific effectors we performed mixed lymphocyte/tumour cultures (MLTC) with autologous or allogeneic tumour cells. No stimulation of cytotoxicity against the autologous tumour was detected after MLTC in nine different TAL populations, using autologous or allogeneic tumours as stimulators. On the contrary, peripheral blood lymphocytes from two patients after MLTC with the autologous tumour showed increased killing of the autologous and decreased killing of an allogeneic target. In conclusion TIL and TAL from ovarian carcinoma expanded in vitro with interleukin-2 usually have non-MHC-restricted cytotoxicity and variable degrees of reactivity against the autologous tumour. A preferential killing for the autologous tumour was not observed even after MLTC. These results do not exclude the existence of tumour-specific cytotoxic T lymphocytes in ovarian carcinoma; nevertheless they suggest that putative specific effectors have very low frequency and that culture techniques for expanding their growth more selectively are still to be optimized.     

Chemo-adoptive immunotherapy of nude mice implanted with human colorectal carcinoma and melanoma cell lines

Summary The antitumor effects of chemotherapy, recombinant human interleukin-2 (IL-2), recombinant human interferon A/D (IFN), allogeneic human lymphokine-activated killer (LAK) cells, and antitumor monoclonal antibody (mAb), administered alone and in various combinations, were tested in athymic nude mice carrying human tumor xenografts. Treatment began 6–18 days after i.v. or i.p. inoculation of colorectal carcinoma or melanoma cell lines, when macroscopic growths were evident. Chemotherapy consisted of two or three courses of 5-fluorouracil (5-FU) or dacarbazine. IL-2 and/or IFN were administered three to five times weekly for 1–3 weeks, usually starting 2–5 days after chemotherapy. Human LAK cells were infused once or twice weekly for 2 or 3 weeks concurrently with IL-2. In some experiments, murine anticolorectal carcinoma mAb (SF25) was administered. In both tumor systems, chemotherapy alone or immunotherapy alone (IL-2, IL-2 + LAK cells, IFN, IL-2 + IFN ± LAK cells) had little or no therapeutic effects. Additive effects were obtained by combining chemotherapy with IL-2 and LAK cells or with IL-2 and IFN. In the majority of the experiments, the most effective combination was chemotherapy + IL-2 + IFN + LAK cells. Treatment with mAb was beneficial in the colorectal carcinoma system when combined with 5-FU + IL-2 or 5-FU + IL-2 + IFN. Homing experiments with radiolabeled human and mouse LAK cells injected i.v. showed increased early accumulation in the liver and lungs, whereas freshly explanted mouse splenocytes localized mostly in the spleen and liver. The tissue distribution pattern of human LAK cells was similar in normal and tumor-bearing mice (with lung metastases). These findings suggest that combination of chemotherapy with cytokines and LAK cells can be partially effective for advanced solid human tumors even in the absence of the host's T-cell immune response. Preliminary experiments showed that tumor-specific, anti-melanoma T-cell clones were effective in local (s.c.) tumor growth inhibition (Winn assay) following coinjection with the autologous tumor cells.     

Effects of α2- and β-adrenoceptor agonists on growth hormone secretion following lesion of the noradrenergic system of the rat

The aim of the present investigation was to lesion the noradrenergic system and to measure the effect on growth hormone (GH) secretion following peripheral administration of ₂- and -adrenoceptor agonists. Direct injection of these agonists into the paraventricular nucleus of the hypothalamus (PVN) and its effect on GH secretion were also investigated. Systemic administration of N-2-chloroethyl-N-ethyl-2-bromobenzylamine (DSP₄, 60 mg/kg, injected i.p. 10 days prior to experimentation) significantly decreased the noradrenaline (NA) content of the hippocampus, frontal cortex and hypothalamus but had no effect on the dopamine (DA) or serotonin (5-HT) content of these areas. Bilateral injection of 6-hydroxydopamine (6-OHDA, 10 g/l, 14 days prior to experimentation) into the medial forebrain bundle (MFB) caused a greater reduction of NA and also decreased the DA and 5-HT content of the hypothalamus. Analysis of the PVN of the hypothalami of rats following 6-OHDA lesion of the MFB showed significantly decreased NA and 5-HT content. Neither DSP₄ treatment nor 6-OHDA lesion of the MFB affected the clonidine (250 g/kg, i.p.) induced stimulation of GH secretion. Injection of isoproterenol (1 mg/kg, i.p.) had varying effects on GH secretion. It stimulated GH release in control rats but not in DSP₄ or MFB lesioned rats. Direct injection of clonidine (0.1 g/l) into the PVN significantly stimulated GH secretion, whereas injection of isoproterenol (2.5 g/l) into the PVN did not affect GH levels when compared to controls. The results of the present study do not support the hypothesis that hypoactivity of the central noradrenergic system may be the cause of the blunted GH response to clonidine observed in depressed patients.     

The “ON”-bipolar agonist,L-2-amino-4-phosphonobutyrate,blocks light-evoked cone contraction in xenopus eye cups

Rhythmic photoreceptor metabolism in relationship to light-dark cycles is now thought be regulated through a retinal feed-back mechanism with dopamine serving as a principal signal initiating light-evoked events. In order to test the hypothesis that depolarizing ON-bipolar neurons participate in the retinal signalling pathway, we determined the effects of L-2-amino-4-phosphonobutyrate (L-APB) on light-evoked cone contraction in eye cups fromXenopus laevis. L-APB blocked the response stereospecifically when applied over a broad concentration range. The high specificity of L-APB in retina suggests that sign-inverting bipolar neurons which depolarize in light are in the signalling pathway. One possibility is that this pathway conveys signals that regulate dopamine release.Special issue dedicated to Dr. Frederick E. Samson.     

4',6-Dichloroflavan (BW683C): Enantiomeric separation by hplc/dad and antirhinovirus activity

Racemic 4',6-dichloroflavan (BW683C), a highly effective inhibitor of rhinovirus serotype 1B in vitro, was resolved by high-performance liquid chromatography on a chiral stationary phase. The enantiomers were separately collected and circular dichroism curves were obtained, in order to determine the absolute configuration of the two enantiomers. The activity of the isomers was studied on human rhinovirus serotype 1B multiplication in HeLa cell cultures, by means of the plaque reduction assay. Both enantiomers were potent inhibitors of virus replication; by comparing the IC₅₀ values, the S form was 3.5 times more effective than the R form.