Mechanism of intestinal formation of deoxycholic acid from cholic acid in humans: evidence for a 3-oxo-delta 4-steroid intermediate

12 alpha-Hydroxy-3-oxo-4-cholenoic acid coupled to an adenosine nucleotide has been shown to be a metabolite of cholic acid in the intestinal anaerobic bacteria, Eubacterium species VPI 12708 (1987. J. Biol. Chem. 262: 4701-4707) and it has been suggested that this may be an intermediate in the conversion of cholic acid into deoxycholic acid. The possibility that the intestinal conversion of cholic acid into deoxycholic acid involves a 3-oxo-delta 4-steroid as an intermediate has been studied in the present work by use of [3 beta-3H]- and [5-3H]-labeled cholic acid. Whole cells as well as cell extracts of Eubacterium sp. VPI 12708 catalyzed conversion of [3 beta-3H] + [24-14C]cholic acid into deoxycholic acid with loss of about 50% of 3H label. When unlabeled chenodeoxycholic acid (20 microM) was added along with [3 beta-3] + [24-14C]cholic acid, then approximately 85% of the [3 beta-3H]-labeled was lost from deoxycholic acid. After administration of the same mixture to two healthy volunteers, deoxycholic acid could be isolated that had lost 81 and 84%, respectively, of the 3H label. Conversion of a mixture of [5-3H]- and [24-14C]labeled cholic acid by the above intestinal bacteria or cell extracts led to loss of 79-94 of the [5-3H] label.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of intestinal 7 alpha-dehydroxylation of cholic acid: evidence that allo-deoxycholic acid is an inducible side-product

We previously reported that the 7 alpha-dehydroxylation of cholic acid appears to be carried out by a multi-step pathway in intestinal anaerobic bacteria both in vitro and in vivo. The pathway is hypothesized to involve an initial oxidation of the 3 alpha-hydroxy group and the introduction of a double bond at C4-C5 generating a 3-oxo-4-cholenoic bile acid intermediate. The loss of water generates a 3-oxo-4,6-choldienoic bile acid which is reduced (three steps) yielding deoxycholic acid. We synthesized, in radiolabel, the following putative bile acid intermediates of this pathway 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acid, 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholanoic acid, 12 alpha-dihydroxy-3-oxo-4,6-choldienoic acid, and 12 alpha-hydroxy-3-oxo-4-cholenoic acid and showed that they could be converted to 3 alpha,12 alpha-dihydroxy-5 beta-cholanoic acid (deoxycholic acid) by whole cells or cell extracts of Eubacterium sp. VPI 12708. During studies of this pathway, we discovered the accumulation of two unidentified bile acid intermediates formed from cholic acid. These bile acids were purified by thin-layer chromatography and identified by gas-liquid chromatography-mass spectrometry as 12 alpha-hydroxy-3-oxo-5 alpha-cholanoic acid and 3 alpha,12 alpha-dihydroxy-5 alpha-cholanoic (allo-deoxycholic acid). Allo-deoxycholic acid was formed only in cell extracts prepared from bacteria induced by cholic acid, suggesting that their formation may be a branch of the cholic acid 7 alpha-dehydroxylation pathway in this bacterium.     

Independently evolving species in asexual bdelloid rotifers

Asexuals are an important test case for theories of why species exist. If asexual clades displayed the same pattern of discrete variation as sexual clades, this would challenge the traditional view that sex is necessary for diversification into species. However, critical evidence has been lacking: all putative examples have involved organisms with recent or ongoing histories of recombination and have relied on visual interpretation of patterns of genetic and phenotypic variation rather than on formal tests of alternative evolutionary scenarios. Here we show that a classic asexual clade, the bdelloid rotifers, has diversified into distinct evolutionary species. Intensive sampling of the genus Rotaria reveals the presence of well-separated genetic clusters indicative of independent evolution. Moreover, combined genetic and morphological analyses reveal divergent selection in feeding morphology, indicative of niche divergence. Some of the morphologically coherent groups experiencing divergent selection contain several genetic clusters, in common with findings of cryptic species in sexual organisms. Our results show that the main causes of speciation in sexual organisms, population isolation and divergent selection, have the same qualitative effects in an asexual clade. The study also demonstrates how combined molecular and morphological analyses can shed new light on the evolutionary nature of species.     

Spermiogenesis in Seison nebaliae (Rotifera, Seisonidea): further evidence of a rotifer-acanthocephalan relationship

The spermatozoa of Seison nebaliae are filiform cells about 70 mum long with a diameter of 0.6 mum. They have a slightly enlarged head, 2.5 mum long, followed by a long cell body. The flagellum starts from the head, and runs parallel to the cell body, contained in a groove along it. The head contains an acrosome, two large, paired para-acrosomal bodies, the basal body of the flagellum and the anterior thin extremity of the nucleus. The cell body contains the main portion of the nucleus, a single mitochondrion located in its distal portion, and many accessory bodies with different shapes. The flagellum has a 9 + 2 axoneme. The study of spermiogenesis shows the Golgian origin of the acrosome and the para-acrosomal bodies and reveals some peculiarities: a folding of the perinuclear cisterna is present between the proacrosome and the basal body of the flagellum in early spermatids and the flagellum runs in a canal inside the spermatid cytoplasm. The basal body migrates anteriorly. These characters are shared partly by the Rotifera Monogononta and, to a large extent, by the Acanthocephala studied so far. Many details of the spermiogenetic process are identical to those of Acanthocephala, thus suggesting that the processes in the two taxa are homologous.     

tBid induces alterations of mitochondrial fatty acid oxidation flux by malonyl-CoA-independent inhibition of carnitine palmitoyltransferase-1

Recent studies suggest a close relationship between cell metabolism and apoptosis. We have evaluated changes in lipid metabolism on permeabilized hepatocytes treated with truncated Bid (tBid) in the presence of caspase inhibitors and exogenous cytochrome c. The measurement of beta-oxidation flux by labeled palmitate demonstrates that tBid inhibits beta-oxidation, thereby resulting in the accumulation of palmitoyl-coenzyme A (CoA) and depletion of acetyl-carnitine and acylcarnitines, which is pathognomonic for inhibition of carnitine palmitoyltransferase-1 (CPT-1). We also show that tBid decreases CPT-1 activity by a mechanism independent of both malonyl-CoA, the key inhibitory molecule of CPT-1, and Bak and/or Bax, but dependent on cardiolipin decrease. Overexpression of Bcl-2, which is able to interact with CPT-1, counteracts the effects exerted by tBid on beta-oxidation. The unexpected role of tBid in the regulation of lipid beta-oxidation suggests a model in which tBid-induced metabolic decline leads to the accumulation of toxic lipid metabolites such as palmitoyl-CoA, which might become participants in the apoptotic pathway.     

A synthetic amino acid substitution of Tyr10 in Aβ peptide sequence yields a dominant negative variant in amyloidogenesis

Alzheimer's disease (AD) is the most common cause of dementia in elderly people, and age is the major nongenetic risk factor for sporadic AD. A hallmark of AD is the accumulation of amyloid in the brain, which is composed mainly of the amyloid beta-peptide (Aβ) in the form of oligomers and fibrils. However, how aging induces Aβ aggregation is not yet fully determined. Some residues in the Aβ sequence seem to promote Aβ-induced toxicity in association with age-dependent risk factors for AD, such as (i) increased GM1 brain membrane content, (ii) altered lipid domain in brain membrane, (iii) oxidative stress. However, the role of Aβ sequence in promoting aggregation following interaction with the plasma membrane is not yet demonstrated. As Tyr10 is implicated in the induction of oxidative stress and stabilization of Aβ aggregation, we substituted Tyr 10 with a synthetic amino acid that abolishes Aβ-induced oxidative stress and shows an accelerated interaction with GM1. This variant peptide shows impaired aggregation properties and increased affinity for GM1. It has a dominant negative effect on amyloidogenesis in vitro, in cellulo, and in isolated synaptosomes. The present study shed new light in the understanding of Aβ-membrane interactions in Aβ-induced neurotoxicity. It demonstrates the relevance of Aβ sequence in (i) Aβ-membrane interaction, underlining the role of age-dependent enhanced GM1 content in promoting Aβ aggregation, (ii) Aβ aggregation, and (iii) Aβ-induced oxidative stress. Our results open the way for the design of peptides aimed to inhibit Aβ aggregation and neurotoxicity.     

Volume and morphology changes of a bdelloid rotifer species (Macrotrachela quadricornifera) during anhydrobiosis

Following a study on the changes occurring in a bdelloid species (Macrotrachela quadricornifera, Rotifera, Bdelloidea) when entering anhydrobiosis, we investigated the changes in morphology, including weight and volume during the transition from the active hydrated to the dormant anhydrobiotic state by scanning electron microscopy, confocal microscopy and light microscopy. We compared sizes and morphologies of hydrated extended, hydrated contracted and anhydrobiotic specimens. Bdelloid musculature is defined: longitudinal muscles are contracted in the hydrated contracted animal (head and foot are retracted inside the trunk), but appear loose in the anhydrobiotic animal. When anhydrobiotic, M. quadricornifera appears much smaller in size, with a volume reduction of about 60% of the hydrated volume, and its internal organization undergoes remarkable modifications. Internal body cavities, clearly distinguishable in the hydrated extended and contracted specimens, are no longer visible in the anhydrobiotic specimen. Concomitantly, M. quadricornifera loses more than 95% of its weight when anhydrobiotic; this is more than expected from the volume reduction data and could indicate the presence of space-filling molecular species in the dehydrated animal. We estimate that the majority of body mass loss and volume reduction can be ascribed to the water loss from the body cavity during desiccation.     

Mesenchymal stromal cells from amniotic fluid are less prone to senescence compared to those obtained from bone marrow: An in vitro study

Mesenchymal stromal cells (MSCs) are considered to be an excellent source in regenerative medicine. They contain several cell subtypes, including multipotent stem cells. MSCs are of particular interest as they are currently being tested using cell and gene therapies for a number of human diseases. They represent a rare population in tissues; for this reason, they require, before being transplanted, an in vitro amplification. This process may induce replicative senescence, thus affecting differentiation and proliferative capacities. Increasing evidence suggests that MSCs from fetal tissues are significantly more plastic and grow faster than MSCs from bone marrow. Here, we compare amniotic fluid mesenchymal stromal cells (AF‐MSCs) and bone marrow mesenchymal stromal cells (BM‐MSCs) in terms of cell proliferation, surface markers, multidifferentiation potential, senescence, and DNA repair capacity. Our study shows that AF‐MSCs are less prone to senescence with respect to BM‐MSCs. Moreover, both cell models activate the same repair system after DNA damage, but AF‐MSCs are able to return to the basal condition more efficiently with respect to BM‐MSCs. Indeed, AF‐MSCs are better able to cope with genotoxic stress that may occur either during in vitro cultivation or following transplantation in patients. Our findings suggest that AF‐MSCs may represent a valid alternative to BM‐MSCs in regenerative medicine, and, of great relevance, the investigation of the mechanisms involved in DNA repair capacity of both AF‐MSCs and BM‐MSCs may pave the way to their rational use in the medical field.