Phenolic 9,10-secosteroids as products of the catabolism of bile acids by a Pseudomonas sp.

The obligate aerobe, Pseudomonas putida ATCC 31752, efficiently utilises bile acids as a source of carbon and energy for growth and maintenance. When aeration is considerably restricted, a consequence to the catabolism of the bile acids in a fermentor is an accumulation of certain steroidal catabolites. Evidence is presented to show that among these are hydroxy-9,10-seco-1,3,5(10)-androstratriene-9,17-diones and those from four of the common bile acids, cholic, chenodeoxycholic, hyodeoxycholic and deoxycholic acids have been isolated and their structures determined. The product of catabolism of hyodeoxycholic acid appears to exist in a hemi-acetal form which readily forms an acetal during isolation procedures. All but one of these are described for the first time.     

Aerobic catabolism of bile acids.

Seventy-eight stable cultures obtained by enrichment on media containing ox bile or a single bile acid were able to utilize one or more bile acids, as well as components of ox bile, as primary carbon sources for growth. All isolates were obligate aerobes, and most (70) were typical (48) or atypical (22) Pseudomonas strains, the remainder (8) being gram-positive actinomycetes. Of six Pseudomonas isolates selected for further study, five produced predominantly acidic catabolites after growth on glycocholic acid, but the sixth, Pseudomonas sp. ATCC 31752, accumulated as the principal product a neutral steroid catabolite. Optimum growth of Pseudomonas sp. ATCC 31752 on ox bile occurred at pH 7 to 8 and from 25 to 30 degrees C. No additional nutrients were required to sustain good growth, but growth was stimulated by the addition of ammonium sulfate and yeast extract. Good growth was obtained with a bile solids content of 40 g/liter in shaken flasks. A near-theoretical yield of neutral steroid catabolites, comprising a major (greater than 50%) and three minor products, was obtained from fermentor growth of ATCC 31752 in 6.7 g of ox bile solids per liter. The possible commercial exploitation of these findings to produce steroid drug intermediates for the pharmaceutical industry is discussed.     

Study on the metabolites of 1alpha,25-dihydroxyvitamin D4

Three major metabolites of 1alpha,25-dihydroxyvitamin D(4) were isolated from the bile of rat and the structures were elucidated on the basis of spectral data and the periodate oxidative cleavage of the diol structures of the metabolites. One of the metabolites was the known calcitroic acid. Another two metabolites were isomers and identified as 9,10-secoergosta-5,7,10(19)-triene-1alpha,3beta,24,25-tetrahydroxy-26-oic acid and 9,10-secoergosta-5,7,10(19)-triene-1alpha,3beta,24,25-tetrahydroxy-28-oic acid. It was found that 1alpha,25-dihydroxyvitamin D(4) is metabolized in a similar manner in vivo to that of 1alpha,25-dihydroxyvitamin D(2) but differently from 1alpha,25-dihydroxyvitamin D(3).     

Detection,isolation and structure elucidation of a chlorophyll a catabolite from autumnal senescent leaves of Cercidiphyllum japonicum

Extracts of autumnal leaves of the dicotyledonous, deciduous tree Cercidiphyllum japonicum cultivated at the Botanical Garden of Fribourg, Switzerland, were screened for chlorophyll catabolites by TLC utilizing the chromic acid degradation test. The constitution of the isolated material was elucidated by spectroscopy. The structure, an optically active bile-pigment-like 19-formyl-1[21H,22H]bilinone derivative, reveals that this compound originates from chlorophyll a and resembles the structures of previously isolated chlorophyll catabolites from the green alga Chlorella protothecoides and from the angiosperms, the monocot Hordeum vulgare and the dicot Brassica napus.     

Heat evolution of microbial catabolism: effects of monooxygenases.

Heat evolved during microbial oxidation, expressed as a fraction of total energy available from each compound, was increased by monooxygenase participation. It was less for growth substrate catabolites than for their isomers.     

Structure elucidation of metabolites of gambogic acid in vivo in rat bile by high-performance liquid chromatography-mass spectrometry and high-performance liquid chromatography-nuclear magnetic resonance

Gambogic acid (GBA), the main component of Gamboge, possesses significant anti-tumour activity. Due to its structural complexity, little is known about GBA metabolism. Here, we investigate the metabolism of GBA in vivo in rat bile. Identification of the metabolites formed was elucidated using high-performance liquid chromatography (HPLC) with UV-vis detection, HPLC/ion trap electrospray ionization-mass spectrometry, as well as HPLC/nuclear magnetic resonance. Four main metabolites were determined. Two phase I metabolites, 10-hydroxygambogic acid and 9,10-epoxygambogic acid, were oxides on the 9,10-olefinic bond of GBA. The others phase II metabolites, were 9,10-epoxygambogic acid-30-O-glucuronide and 10-hydroxylgambogic acid-30-O-glucuronide.     

Bile acid catabolites accumulated by transposon-induced mutants ofPseudomonas putida: Production of hydroxylated 1,4-androstadiene-3,17-diones

Summary Transposon mutagenesis of a bile acid-utilizingPseudomonas putida strain generated 5 classes of mutants based on their ability to accumulate steroild catabolites. Bile acids (up to 5%) could be fermented using mutant strains, to the appropriate hydroxy-1,4-androstadiene-3,17-dione type product in yields close to theoretical.     

Identification of two pigments and a hydroxystilbene antibiotic from Photorhabdus luminescens.

Two yellow pigments were isolated for the first time from the entomopathogenic bacterium Photorhabdus luminescens in liquid culture and were identified as the anthraquinone derivatives 3,8-dimethoxy-1-hydroxy-9,10-anthraquinone (minor) and 1,3-dimethoxy-8-hydroxy-9,10-anthraquinone (major). A known antibiotic, 3,5-dihydroxy-4-isopropylstilbene, was also detected and for the first time showed strong fungicidal activity against several fungi of medical and agricultural importance.     

Identification of Catabolites of Chlorophyll-Porphyrin in Senescent Rape Cotyledons

Developing shoots of rape seedlings (Brassica napus L.) were excised and fed with 4-[14C]5-aminolevulinic acid to label the pyrroles in chlorophyll (Chl) synthesized during the final phase of expansion and greening of the cotyledons. About 80% of 14C taken up into the cotyledons was incorporated into Chl. The subsequent incubation of labeled shoots in permanent darkness caused the rapid loss of labeled Chl while increasing proportions of 14C appeared in the fraction of water-soluble compounds. Reversed-phase high performance liquid chromatography resolved three nonfluorescent polar catabolites of Chl-porphyrin that were progressively accumulated as senescence advanced. At intermediate stages of senescence, the cotyledons contained a fluorescent radio-active derivative of Chl that was also detectable, together with traces of other putative fluorescent catabolites, in isolated senescent chloroplasts. The nonfluorescent catabolites, identified by means of radiolabeling, were also found to accumulate in attached cotyledons senescing under photoperiod; under these conditions, one of the compounds, NCC-1, was particularly abundant. The catabolites of rape exhibited the same ultraviolet spectra, characterized by a maximum at 320 nm, as a previously reported secoporphinoid catabolite from barley (B. Krautler, B. Jaun, W. Amrein, K. Bortlik, M. Schellenberg, P. Matile [1992] Plant Physiol Biochem 30: 333-346). Different polarities suggest, however, that the structures may be different. A terminology for Chl catabolites is proposed because present knowledge suggests that a large number of different structures results from species-specific processing of breakdown products and may require a suitable nomenclature.     

肠道菌群与胆汁酸的互作在 相关疾病中的作用

胆汁酸在人体的胆固醇代谢、脂质消化、宿主-微生物相互作用及通路调控等方面具有重要作用。大多数胆汁酸(95%)通过肝肠循环重回收,还有约5%作为结肠内细菌生物转化的基质。胆汁酸微生物转化中涉及的各种酶可通过肠道细菌培养而被验证,证明其有种属特异性。最近,生物信息学方法揭示了这些酶有多种亚型。因此,在胆汁酸转化中肠道菌群发挥重要的作用,微生物群落结构和功能对次级胆汁酸在胆汁酸池中的分布有深刻影响。研究认为胆汁酸和胆汁酸池的组成与几种疾病有关,包括炎症性肠病、代谢综合征和结直肠癌。最近,人们的重点放在肠道菌群如何改变胆汁酸进而导致或减轻某些疾病。本文总结了肠道菌群、胆汁酸生物转化和疾病状态之间的相互作用的研究进展。     

Chlorophyll Breakdown in Senescent Chloroplasts (Cleavage of Pheophorbide a in Two Enzymic Steps)

The cleavage of pheophorbide (Pheide) a into primary fluoescent chlorophyll (Chl) catabolites (pFCCs) in senescent chloroplasts was investigated. Chloroplast preparations isolated from senescent canola (Brassica napus) cotyledons exhibited light-dependent production of pFCC when assay mixtures were supplemented with ferredoxin (Fd). pFCC production in detergent-solubilized membranes was dependent on the presence of an Fd-reducing system. Pheide a cleavage required the action of two proteins, Pheide a oxygenase and a stroma protein. In the absence of stroma protein, Pheide a oxygenase converted Pheide a into a red Chl catabolite (RCC), the presumptive intermediary product of Pheide a cleavage. Incubation of the stroma protein (RCC reductase) together with chemically synthesized RCC resulted in the production of three different FCCs. Two of these catabolites were identical to the pFCCs from canola or barley (Hordeum vulgare) (pFCC-1) and sweet pepper (Capsicum annuum) (pFCC-2), respectively. Thus, the conversion of Pheide a to pFCC could be demonstrated to proceed in two consecutive steps, and both reactions depended on reduced Fd as the source of electrons. The function of Fd in Chl breakdown in vivo is corroborated by the marked retention of this protein until the late stages of senescence, as demonstrated by immunoblotting.     

Transcriptome atlas of eight liver cell types uncovers effects of histidine catabolites on rat liver regeneration

Eight liver cell types were isolated using the methods of Percoll density gradient centrifugation and immunomagnetic beads to explore effects of histidine catabolites on rat liver regeneration. Rat Genome 230 2.0 Array was used to detect the expression profiles of genes associated with metabolism of histidine and its catabolites for the above-mentioned eight liver cell types, and bioinformatic and systems biology approaches were employed to analyse the relationship between above genes and rat liver regeneration. The results showed that the urocanic acid (UA) was degraded from histidine in Kupffer cells, acts on Kupffer cells itself and dendritic cells to generate immune suppression by autocrine and paracrine modes. Hepatocytes, biliary epithelia cells, oval cells and dendritic cells can convert histidine to histamine, which can promote sinusoidal endothelial cells proliferation by GsM pathway, and promote the proliferation of hepatocytes and biliary epithelia cells by GqM pathway.     

Structure of oxoflavidin,a 9,10-dihydrophenanthropyrone from Coelogyne elata

Oxoflavidin, a new phenolic compound isolated from the Himalayan orchid Coelogyne elata was shown to be 2,7-dihydroxy-9,10-dihydro-5H-phenanthro[4,5-bcd]pyran-5-one (2d) by spectral and chemical evidence.     

Novel assay for determining the metabolic fate of juvenile hormone III: A study with Drosophila melanogaster

A thin-layer chromatographic assay was developed for the resolution of hydrolytic and conjugative catabolites of juvenile hormone (JH). A single-dimension, dual-development thin-layer system allowed complete resolution of the catabolites. Thus, this system provided a means for the rapid and economic analysis of JH hydrolysis even when different hydrolytic activities were present concurrently. Purified hydrolytic enzymes were found to be superior to chemical methods for the generation of small amounts of standards of JH catabolites. The relative levels of activities of an epoxide hydrolase and an esterase toward JH III were found to be similar in microsomal preparations from three lines of adult Drosophila melanogaster isolated from a field population. However, selection of flies by exposure to cut orange resulted in the elevation of levels of epoxide hydrolase activities, whereas esterase levels were not affected to the same extent. The formation of the JH acid-diol was not detected under the conditions of this study, suggesting that the JH acid and diol were not good substrates for epoxide hydrolase and juvenile hormone esterase, respectively.     

Human IgA as a heterovalent ligand: switching from the asialoglycoprotein receptor to secretory component during transport across the rat hepatocyte

Asialoglycoproteins are taken up by the rat liver for degradation; rat polymeric IgA is taken up via a separate receptor, secretory component (SC), for quantitative delivery to bile. There is negligible uptake of these ligands by the converse receptor, and only a low level of missorting of ligands to opposite destinations. The two pathways are not cross-inhibitable and operate independently (Schiff, J.M., M. M. Fisher, and B. J. Underdown, 1984, J. Cell Biol., 98:79-89). We report here that when human IgA is presented as a ligand in the rat, it is processed using elements of both pathways. To study this in detail, different IgA fractions were prepared using two radiolabeling methods that provide separate probes for degradation or re-secretion. Behavior of intravenously injected human polymeric IgA in the rat depended on its binding properties. If deprived of SC binding activity by affinity adsorption or by reduction and alkylation, greater than 80% of human IgA was degraded in hepatic lysosomes; radioactive catabolites were released into bile by a leupeptin-inhibitable process. If prevented from binding to the asialoglycoprotein receptor by competition or by treatment with galactose oxidase, human IgA was cleared and transported to bile directly via SC, but its uptake was about fivefold slower than rat IgA. Untreated human IgA was taken up rapidly by the asialoglycoprotein receptor, but depended on SC binding to get to bile: the proportion secreted correlated 1:1 with SC binding activity determined in vitro, and the IgA was released into bile with SC still attached. These results demonstrate that human IgA is normally heterovalent: it is first captured from blood by the asialoglycoprotein receptor, but escapes the usual fate of asialoglycoproteins by switching to SC during transport. Since the biliary transit times of native human and rat IgA are the same, it is probable that the receptor switching event occurs en route. This implies that the two receptors briefly share a common intracellular compartment.     

AtMRP2, an Arabidopsis ATP binding cassette transporter able to transport glutathione S-conjugates and chlorophyll catabolites: functional comparisons with Atmrp1.

Three ATP binding cassette (ABC) transporter-like activities directed toward large amphipathic organic anions have recently been identified on the vacuolar membrane of plant cells. These are the Mg-ATP-energized, vanadate-inhibitable vacuolar accumulation of glutathione S-conjugates (GS conjugates), chlorophyll catabolites, and bile acids, respectively. Although each of these activities previously had been assigned to distinct pumps in native plant membranes, we describe here the molecular cloning, physical mapping, and heterologous expression of a gene, AtMRP2, from Arabidopsis thaliana that encodes a multispecific ABC transporter competent in the transport of both GS conjugates and chlorophyll catabolites. Unlike its isoform, AtMRP1, which transports the model Brassica napus chlorophyll catabolite transporter substrate Bn-NCC-1 at low efficiency, heterologously expressed AtMRP2 has the facility for simultaneous high-efficiency parallel transport of GS conjugates and Bn-NCC-1. The properties of AtMRP2 therefore establish a basis for the manipulation of two previously identified plant ABC transporter activities and provide an explanation for how the comparable transporter in native plant membranes would be systematically mistaken for two distinct transporters. These findings are discussed with respect to the functional organization of AtMRP2, the inability of AtMRP2 and AtMRP1 to transport the model bile acid transporter substrate taurocholate (despite the pronounced sensitivity of both to direct inhibition by this agent), the differential patterns of expression of their genes in the intact plant, and the high capacity of AtMRP2 for the transport of glutathionated herbicides and anthocyanins.     

Receptor-mediated biliary transport of immunoglobulin A and asialoglycoprotein: sorting and missorting of ligands revealed by two radiolabeling methods

In the rat, all receptor-bindable immunoglobulin A (IgA), and 1-4% of injected asialoglycoprotein (ASG), are transported from blood to bile intact. The major fraction of the ASG is degraded in hepatic lysosomes. The study described here was designed to elucidate the sorting that occurs in hepatocytes subsequent to receptor binding of ligands not sharing the same fate. We show that conjugation of protein with the Bolton and Hunter reagent can be used as a probe for the lysosomal pathway, since 50% of the reagent is released into bile after lysosomal degradation of internalized protein. Radiolabeling by iodine monochloride was alternatively used to follow the direct pathways that deliver intact IgA and ASG to bile. After intravenous injection of labeled proteins, first intact ASG and IgA, and then radioactive catabolites from degraded protein, were released into bile. No proteolytic intermediates were detected, and the transport of IgA or ASG directly to bile was not affected by the lysosomal protease inhibitor leupeptin. These observations indicate that divergence of the direct biliary transport pathways from the degradation pathway occurs at a stage preceding delivery to lysosomes, possibly at the cell surface. Competition studies showed that all three pathways (including the biliary transport of intact ASG) are receptor mediated, but even at supersaturating doses the uptake and processing of IgA and ASG occur independently. We propose that IgA and ASG receptors are not frequently in juxtaposition on the plasma membrane, but that ASG, after binding to its receptor, is occasionally missorted into the biliary transport pool.     

Release of Photosynthetic Protein Catabolites by Blebbing from Thylakoids

Thylakoid proteins and their catabolites have been detected in lipid-protein particles isolated from the stroma of intact chloroplasts obtained from primary leaves of 2-week-old bean seedlings (Phaseolus vulgaris L. cv Kinghorn). The lipid-protein particles bear morphological resemblance to plastoglobuli seen in the chloroplasts of senescing leaves, but they are much smaller. They range from 10 to 320 nm in radius, are uniformly stained in thin sections visualized by transmission electron microscopy, and are discernible in the stroma of chloroplasts in corresponding thin-sectioned leaf tissue. The lipid-protein particles contain thylakoid lipids and are enriched in free fatty acids. Specifically, the free-to-esterified fatty acid ratio is about 1:1 in the particles compared to only 1:18 for corresponding thylakoid membranes. Western blot analyses indicate that these particles also contain thylakoid proteins and, in some cases, catabolites of these proteins including the CF1 [beta] and [gamma] subunits of ATPase, cytochrome f, and the 31- and 33-kD proteins of PSII. Lipid-protein particles with similar properties were generated in vitro from isolated, light-stressed thylakoids. Collectively, these data suggest that blebbing of lipid-protein particles may be a means of removing potentially destabilizing macromolecular catabolites from thylakoid membrane bilayers.     

Effect of various chemicals on the metabolism of benzo(a)pyrene by cultured rat colon

The effect of various co- and anti-carcinogens of colon carcinogenesis on the metabolism of benzo(a)pyrene (BP) in cultured rat colon is reported. Rat colon enzymatically converted BP into metabolites which bind to cellular macromolecules i.e., DNA and protein. Activity of aryl hydrocarbon hydroxylase (AHH) activity and binding levels of BP to macromolecules were higher in the descending colon when compared to other segments. The major metabolites of BP, extractable with ethylacetate, were quinones, tetrols, 7,8-diol and a peak containing 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene and 7,8,9-trihydroxy-7,8-dihydrobenzo(a)pyrene. The binding levels of BP to DNA and protein in the explant was lowered by co-incubation with 7,8-benzoflavone (7,8-BF) (3.6 and 18.0 μM), a known inhibitor of AHH, and with disulfiram (100 μM), an anti-oxidant. The absence of vitamin A in the media also resulted in a lower level of BP binding to DNA and protein and in lower activity of AHH. Pretreatment with known inducers of AHH such as phenobarbital (PB) or benz(a)anthracene (BA), did not have any significant effect on the binding levels of BP to DNA or on the AHH activity. of the bile acids investigated only taurodeoxycholic acid significantly increased the binding level of BP to DNA.