Bile Acids: Chemistry, Pathochemistry, Biology, Pathobiology, and Therapeutics

Bile acids and bile alcohols in the form of their conjugates are amphipathic end products of cholesterol metabolism with multiple physiological functions. The great variety of bile acids and bile alcohols that are present in vertebrates are tabulated. Bile salts have an enterohepatic circulation resulting from efficient vectorial transport of bile salts through the hepatocyte and the ileal enterocyte; such transport leads to the accumulation of a pool of bile salts that cycles between the liver and intestine. Bile salt anions promote lipid absorption, enhance tryptic cleavage of dietary proteins, and have antimicrobial effects. Bile salts are signaling molecules, activating nuclear receptors in the hepatocyte and ileal enterocyte, as well as an increasing number of G-protein coupled receptors. Bile acids are used therapeutically to correct deficiency states, to decrease the cholesterol saturation of bile, or to decrease the cytotoxicity of retained bile acids in cholestatic liver disease.     

Myosin I: From yeast to human

Myosin I is a non-filamentous, single-headed, actin-binding motor protein and is present in a wide range of species from yeast to man. The role of these class I myosins have been studied extensively in simple eukaryotes, showing their role in diverse processes such as actin cytoskeleton organization, cell motility, and endocytosis. Recently, studies in metazoans have begun to reveal more specialized functions of myosin I. It will be a major challenge in the future to examine the physiological functions of each class I myosin in different cell types of metazoans.     

Regulation of muscle creatine kinase by phosphorylation in normal and diabetic hearts

Protein kinase C (PKC) is an important signaling molecule in the heart, but its targets remain unclear. Using a PKC substrate antibody, we detected a 40-kDa phosphorylated cardiac protein that was subsequently identified by tandem mass spectroscopy as muscle creatine kinase (M-CK) with phosphorylation at serine 128. The forward reaction using ATP to generate phosphocreatine was reduced, while the reverse reaction using phosphocreatine to generate ATP was increased following dephosphorylation of immunoprecipitated M-CK with protein phosphatase 2A (PP2A) or PP2C. Despite higher PKC levels in diabetic hearts, decreased phosphorylation of M-CK was more prominent than the reduction in its expression. Changes in CK activity in diabetic hearts were similar to those found following dephosphorylation of M-CK from control hearts. The decrease in phosphorylation may act as a compensatory mechanism to maintain CK activity at an appropriate level for cytosolic ATP regeneration in the diabetic heart. Received 15 September 2008; received after revision 30 September 2008; accepted 13 October 2008     

Gene expression in spermiogenesis

Germ cells convey parental genes to the next generation, and only germ cells perform meiosis, which is a mechanism that preserves the parental genes. The fusion of the products of germ cell meiosis, the haploid sperm and egg, creates the next generation. Sperm are the haploid germ cells that contribute genes to the egg. In preparation for this, the haploid round spermatids produced by meiosis undergo drastic morphological changes to become sperm. During this process of spermiogenesis, the nuclear form of the haploid germ cell takes shape, the mitochondria are rearranged in a specific manner, the flagellum develops and the acrosome forms. Spermatogenesis is supported by precise and orderly regulation of gene expression during the changes in chromatin structure, when protamine replaces histone. In this report, we summarize the molecular mechanisms involved in spermiogenesis.Received 2 September 2004; received after revision 7 October 2004; accepted 7 October 2004     

Lysophosphatidic acid up-regulates vascular endothelial growth factor-C and lymphatic marker expressions in human endothelial cells

Lysophosphatidic acid (LPA) is a low-molecular-weight lipid growth factor, which binds to G-protein-coupled receptors. Previous studies have shown that LPA enhances vascular endothelial growth factor-A (VEGF-A) expression in cancer cells and promotes angiogenesis process. However, the roles of LPA in lymphatic vessel formation and lymphangiogenesis have not been investigated. Here, we demonstrated that LPA up-regulated VEGF-C mRNA and protein expressions in human umbilical vein endothelial cells (HUVECs). Furthermore, the expression levels of lymphatic markers, including Prox-1, LYVE-1 and podoplanin, were enhanced in LPA-stimulated tube forming endothelial cells in vitro and in vivo. Moreover, we showed that pretreatment with MAZ51, a VEGFR-3 kinase inhibitor, and introduction of VEGFR-3 siRNA suppressed LPA-induced HUVEC tube formation and lymphatic marker expressions. These results demonstrated that LPA enhances expression of lymphatic markers through activating VEGF-C receptors in endothelial cells. This study provides basic information that LPA might be a target for therapeutics against lymphangiogenesis and tumor metastasis.     

The mode of antifungal action of plant, insect and human defensins

Defensins are small (~5 kDa), basic, cysteine-rich antimicrobial peptides that fulfill an important role in the innate immunity of their host by combating pathogenic invading micro-organisms. Defensins can inhibit the growth or virulence of microorganisms directly or can do so indirectly by enhancing the host's immune system. Because of their wide distribution in nature, defensins are believed to be ancient molecules with a common ancestor that arose more than a billion years ago. This review summarizes current knowledge concerning the mode of antifungal action of plant, insect and human defensins.     

Regulation of chondrocyte differentiation by ADAMTS-12 metalloproteinase depends on its enzymatic activity

ADAMTS-12, a metalloproteinase that belongs to ADAMTS family, is strongly upregulated during chondrogenesis and demonstrates prominent expression in the growth plate chondrocytes. ADAMTS-12 potently inhibits chondrocyte differentiation, as revealed by altered expression of both early and later genes critical for chondrogenesis. In addition, ADAMTS-12-mediated inhibition of chondrogenesis depends on its enzymatic activity, since its point mutant lacking enzymatic activity completely loses this activity. Furthermore, the C-terminal four thrombospondin motifs known to bind COMP substrate is necessary for its full proteolytic activity and inhibition of chondrocyte differentiation. Mechanism studies demonstrate that ADAMTS-12 induces PTHrP, whereas it inhibits IHH during chondrogenesis. Furthermore, PTHrP induces ADAMTS-12 and ADAMTS-12 is hardly detectable in PTHrP-/-growth plate chondrocytes. Importantly, knocking down ADAMTS-12 mRNA levels or blocking ADAMTS-12 activity almost abolishes the PTHrP-mediated inhibition of type X collagen expression. Collectively, these findings demonstrate that ADAMTS-12, a downstream molecule of PTHrP signaling, is a novel regulator of chondrogenesis. X. H. Bai, D.W. Wang: These two authors contributed equally to this work.     

The Role of the Agouti-Related Protein in Energy Balance Regulation

The Agouti-Related Protein (AgRP) is a powerful orexigenic peptide that increases food intake when ubiquitously overexpressed or when administered centrally. AgRP-deficiency, on the other hand, leads to increased metabolic rate and a longer lifespan when mice consume a high fat diet. In humans, AgRP polymorphisms have been consistently associated with resistance to fatness in Blacks and Whites and resistance to the development of type-2 diabetes in African Blacks. Systemically administered AgRP accumulates in the liver, the adrenal gland and fat tissue while recent findings suggest that AgRP may also have inverse agonist effects, both centrally and peripherally. AgRP could thus modulate energy balance via different actions. Its absence or reduced functionality may offer a benefit both in terms of bringing about negative energy balance in obesigenic environments, as well as leading to an increased lifespan.     

Functions and transport of silicon in plants

Silicon exerts beneficial effects on plant growth and production by alleviating both biotic and abiotic stresses including diseases, pests, lodging, drought, and nutrient imbalance. Recently, two genes (Lsi1 and Lsi2) encoding Si transporters have been identified from rice. Lsi1 (low silicon 1) belongs to a Nod26-like major intrinsic protein subfamily in aquaporin, while Lsi2 encodes a putative anion transporter. Lsi1 is localized on the distal side of both exodermis and endodermis in rice roots, while Lsi2 is localized on the proximal side of the same cells. Lsi1 shows influx transport activity for Si, while Lsi2 shows efflux transport activity. Therefore, Lsi1 is responsible for transport of Si from the external solution to the root cells, whereas Lsi2 is an efflux transporter responsible for the transport of Si from the root cells to the apoplast. Coupling of Lsi1 with Lsi2 is required for efficient uptake of Si in rice. Received 21 December 2007; received after revision 29 April 2008; accepted 15 May 2008     

Role of full-length osteoprotegerin in tumor cell biology

Osteoprotegerin (OPG) is a soluble tumor necrosis factor receptor family member, which potently inhibits RANKL-mediated osteoclastogenesis. Numerous constructs have been created for therapeutic purposes in which the heparin-binding and death homology domains of OPG were removed and the remaining peptide (amino acids 22–194) was fused to the Fc domain of human IgG1 (OPG-Fc). The administration of OPG-Fc efficiently counteracted bone loss in a variety of preclinical models of cancers. However, several in vitro studies have shown that native or recombinant full-length OPG not only neuralizes RANKL, but also the death-inducing ligand TRAIL, suggesting that OPG might potentially counteract the anti-tumor activity of TRAIL. Additional evidence suggests that full-length OPG possesses RANKL- and TRAIL-independent biological properties, mainly related to the promotion of endothelial cell survival and angiogenesis. Finally, breast tumor cells overexpressing OPG have shown increased bone metastatic potential in vivo. The relevance of these apparently conflicting findings in tumor cell biology is highlighted. Received 2 September 2008; received after revision 29 September 2008; accepted 13 October 2008     

Hydroxylation of demethoxy-Q6 constitutes a control point in yeast coenzyme Q6 biosynthesis

Coenzyme Q is a lipid molecule required for respiration and antioxidant protection. Q biosynthesis in Saccharomyces cerevisiae requires nine proteins (Coq1p–Coq9p). We demonstrate in this study that Q levels are modulated during growth by its conversion from demethoxy-Q (DMQ), a late intermediate. Similar conversion was produced when cells were subjected to oxidative stress conditions. Changes in Q₆/DMQ₆ ratio were accompanied by changes in COQ7 gene mRNA levels encoding the protein responsible for the DMQ hydroxylation, the penultimate step in Q biosynthesis pathway. Yeast coq null mutant failed to accumulate any Q late biosynthetic intermediate. However, in coq7 mutants the addition of exogenous Q produces the DMQ synthesis. Similar effect was produced by over-expressing ABC1/COQ8. These results support the existence of a biosynthetic complex that allows the DMQ₆ accumulation and suggest that Coq7p is a control point for the Q biosynthesis regulation in yeast. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 04 September 2008; received after revision 22 October 2008; accepted 23 October 2008     

The chemical versatility of the β–α–β fold: Catalytic promiscuity and divergent evolution in the tautomerase superfamily

Tautomerase superfamily members have an amino-terminal proline and a β–α–β fold, and include 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), trans- and cis-3-chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively), malonate semialdehyde decarboxylase (MSAD), and macrophage migration inhibitory factor (MIF), which exhibits a phenylpyruvate tautomerase (PPT) activity. Pro-1 is a base (4-OT, CHMI, the PPT activity of MIF) or an acid (CaaD, cis-CaaD, MSAD). Components of the catalytic machinery have been identified and mechanistic hypotheses formulated. Characterization of new homologues shows that these mechanisms are incomplete. 4-OT, CaaD, cis-CaaD, and MSAD also have promiscuous activities with a hydratase activity in CaaD, cis-CaaD, and MSAD, PPT activity in CaaD and cis-CaaD, and CaaD and cis-CaaD activities in 4-OT. The shared promiscuous activities provide evidence for divergent evolution from a common ancestor, give hints about mechanistic relationships, and implicate catalytic promiscuity in the emergence of new enzymes. Received 22 May 2008; received after revision 20 June 2008; accepted 02 July 2008     

Progesterone inhibits human endothelial cell proliferation through a p53-dependent pathway

Previous studies have shown that progesterone inhibits endothelial cell proliferation through a nuclear receptor-mediated mechanism. Here, we further demonstrate that progesterone at physiologic levels (5 – 500 nM) dose- and time-dependently inhibited DNA synthesis of cultured human umbilical vein endothelial cells (HUVEC). The mRNA and protein levels of p21, p27, and p53 in HUVEC were increased by progesterone. The formation of CDK2-p21 and CDK2-p27 were increased and the CDK2 activity was decreased in the progesterone-treated HUVEC. The progesterone-inhibited [3H]thymidine incorporation was completely blocked when the expressions of p21 and p27 were knocked-down together. Transfection of HUVEC with dominant negative p53 cDNA prevented the progesterone-induced increases in p21 and p27 promoter activity and protein level, decreases in thymidine incorporation, and capillary-like tube formation. Matrigel angiogenesis assay in mice demonstrated the antiangiogenic effect of progesterone in vivo. These findings demonstrate for the first time that progesterone inhibited endothelial cell proliferation through a p53-dependent pathway. Received 28 July 2008; received after revision 25 September 2008; accepted 26 September 2008     

Dissection of the molecular mechanisms that control the nuclear accumulation of transport factors importin-α and CAS in stressed cells

The physiological state of eukaryotic cells controls nuclear trafficking of numerous cargos. For example, stress results in the inhibition of classical protein import, which is characterized by the redistribution of several transport factors. As such, importin-alpha and cellular apoptosis susceptibility protein (CAS) accumulate in nuclei of heat-shocked cells; however, the mechanisms underlying this relocation are not fully understood. We now show that heat upregulates the initial docking of importin-alpha at the nuclear envelope and stimulates the translocation of CAS into the nuclear interior. Moreover, heat exposure compromises the exit of importin-alpha from nuclei and drastically increases its retention in the nucleoplasm, whereas CAS nuclear exit and retention are less affected. Taken together, our results support the idea that heat shock regulates importin-alpha and CAS nuclear accumulation at several levels. The combination of different stress-induced changes leads to the nuclear concentration of both transport factors in heat-stressed cells.     

Effects of ulapualide A and synthetic macrolide analogues on actin dynamics and gene regulation

Several marine macrolide toxins act as potent and specific actin-severing molecules. Recent elucidation of their stereochemistries and modes of interaction with actin has allowed the syntheses of bioactive analogues. Here we used synthetic analogues in a structure-function analysis of ulapualide A, a trisoxazole-based macrolide. Ulapualide A harboured potent actin-depolymerising activity both in cells and in vitro. Its synthetic diastereoisomer was three orders of magnitude less active than the natural toxin and synthetic macrolide fragments lacked actin-capping/ severing activity altogether. Modulation of serum response factor (SRF)-dependent gene expression, as described for other actin-binding toxins, was also examined. Specific changes in response to ulapualide A were not observed, primarily due to its profound effects on cytoskeletal integrity and cell adhesion. Several synthetic fragments of ulapualide A also had no effect on SRF-dependent gene expression. However, inhibition was observed with a molecule corresponding to the extended aliphatic side chain of halichondramide, a structurally related macrolide. These findings indicate that side-chain derivatives of trisoxazole-based macrolides may serve to uncouple gene-regulatory events from actin dynamics. E. Vincent and J. Saxton: These two authors contributed equally Received 27 September 2006; received after revision 30 November 2006; accepted 8 January 2007     

Histone methylation and ubiquitination with their cross-talk and roles in gene expression and stability

Methylation of lysine residues of histones is associated with functionally distinct regions of chromatin, and, therefore, is an important epigenetic mark. Over the past few years, several enzymes that catalyze this covalent modification on different lysine residues of histones have been discovered. Intriguingly, histone lysine methylation has also been shown to be cross-regulated by histone ubiquitination or the enzymes that catalyze this modification. These covalent modifications and their cross-talks play important roles in regulation of gene expression, heterochromatin formation, genome stability, and cancer. Thus, there has been a very rapid progress within past several years towards elucidating the molecular basis of histone lysine methylation and ubiquitination, and their aberrations in human diseases. Here, we discuss these covalent modifications with their cross-regulation and roles in controlling gene expression and stability. Received 24 September 2008; received after revision 21 November 2008; accepted 28 November 2008