ent-Rosane and abietane diterpenoids as cancer chemopreventive agents

Two ent-rosane- (cuzcol, 1 and 6-dehydroxycuzcol, 2) and a abietatriene- (salvadoriol, 3) type diterpenoids have been isolated from Maytenus cuzcoina and Crossopetalum uragoga, respectively, along with five known diterpene compounds (4-8). Their stereostructures have been elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques, and computational data. The absolute configuration of cuzcol was determined by application of Riguera ester procedure. This is the first instance of isolation of ent-rosane diterpenoids from species of the Celastraceae. The isolated diterpenes were found to be potent anti-tumour-promoter agents, and carnosol (7) also showed a remarkable chemopreventive effect in an in vivo two-stage carcinogenesis model.     

Steroid hormones as bactericidal agents to Helicobacter pylori

Helicobacter pylori is a unique bacterial species that assimilates various steroids as membrane lipid components. Our group has recently found, however, that certain steroids may impair the viability of H. pylori. In this study, we go on to reveal that estradiol, androstenedione, and progesterone (PS) all have the potential to inhibit the growth of H. pylori. Of these three steroid hormones, progesterone demonstrated the most effective anti-H. pylori action. 17α-hydroxyprogesterone caproate (17αPSCE), a synthetic progesterone derivative, had a much stronger anti-H. pylori action than progesterone, whereas 17α-hydroxyprogesterone, a natural progesterone derivative, completely failed to inhibit the growth of the organism. Progesterone and 17αPSCE were both found to kill H. pylori through their bacteriolytic action. Among five bacterial species investigated, H. pylori was the only species susceptible to the bactericidal action of progesterone and 17αPSCE. The other four species, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epiderimidis, all resisted this action. Progesterone and free-cholesterol (FC) obstructed each other's effects against the H. pylori cell. Taken in sum, these results suggest that progesterone and FC may bind to the identical region on the H. pylori cell surface. We expect these findings to contribute to the development of a novel anti-H. pylori steroidal agent.     

Estrogen deficiency worsens steatohepatitis in mice fed high-fat and high-cholesterol diet

Recent studies indicate an accelerated progression of nonalcoholic steatohepatitis (NASH) in postmenopausal women. Hypercholesterolemia, an important risk factor for NASH progression, is often observed after menopause. This study examined the effects of estrogen on NASH in ovariectomized (OVX) mice fed a high-fat and high-cholesterol (HFHC) diet. To investigate the effects of estrogen deficiency, OVX mice and sham-operated (SO) mice were fed normal chow or HFHC diet for 6 wk. Next, to investigate the effects of exogenous estrogen replenishment, OVX mice fed with HFHC diet were treated with implanted hormone release pellets (containing 17β-estradiol or placebo vehicle) for 6 wk. OVX mice on the HFHC diet showed enhanced liver injury with increased liver macrophage infiltration and elevated serum cholesterol levels compared with SO-HFHC mice. Hepatocyte monocyte chemoattractant protein-1 (MCP1) protein expression in OVX-HFHC mice was also enhanced compared with SO-HFHC mice. In addition, hepatic inflammatory gene expressions, including monocytes chemokine (C-C motif) receptor 2 (CCR2), were significantly elevated in OVX-HFHC mice. Estrogen treatment improved serum cholesterol levels, liver injury, macrophage infiltration, and inflammatory gene expressions in OVX-HFHC mice. Moreover, the elevated expression of liver CCR2 and MCP1 were decreased by estrogen treatment in OVX-HFHC mice, whereas low-density lipoprotein dose dependently enhanced CCR2 expression in THP1 monocytes. Our study demonstrated that estrogen deficiency accelerated NASH progression in OVX mice fed HFHC diet and that this effect was improved by estrogen therapy. Hypercholesterolemia in postmenopausal women would be a potential risk factor for NASH progression.     

Intestinal SR-BI is upregulated in insulin-resistant states and is associated with overproduction of intestinal apoB48-containing lipoproteins

Intestinal lipid dysregulation is a common feature of insulin-resistant states. The present study investigated alterations in gene expression of key proteins involved in the active absorption of dietary fat and cholesterol in response to development of insulin resistance. Studies were conducted in two diet-induced animal models of insulin resistance: fructose-fed hamster and high-fat-fed mouse. Changes in the mRNA abundance of lipid transporters, adenosine triphosphate cassette (ABC) G5, ABCG8, FA-CoA ligase fatty acid translocase P4, Niemann-Pick C1-Like1 (NPC1L1), fatty acid transport protein 4 (FATP4), and Scavenger Receptor Class B Type I (SR-BI), were assessed in intestinal fragments (duodenum, jejunum, and ileum) using quantitative real-time PCR. Of all the transporters evaluated, SR-B1 showed the most significant changes in both animal models examined. A marked stimulation of SR-B1 expression was observed in all intestinal segments examined in both insulin-resistant animal models. The link between SR-BI expression and intestinal lipoprotein production was then examined in the Caco-2 cell model. SR-B1 overexpression in Caco-2 cells increased apolipoprotein B (apoB) 100 and apoB48 secretion, whereas RNAi knock down of SR-B1 decreased secretion of both apoB100 and apoB48. We also observed changes in subcellular distribution of SR-B1 in response to exogenous lipid and insulin. Confocal microscopy revealed marked changes in SR-BI subcellular distribution in response to both exogenous lipids (oleate) and insulin. In summary, marked stimulation of intestinal SR-BI occurs in vivo in animal models of diet-induced insulin resistance, and modulation of SR-BI in vitro regulates production of apoB-containing lipoprotein particles. We postulate that apical and/or basolateral SR-BI may play an important role in intestinal chylomicron production and may contribute to chylomicron overproduction normally observed in insulin-resistant states.     

Starch biosynthesis in rice endosperm requires the presence of either starch synthase I or IIIa

Starch synthase (SS) I and IIIa are the first and second largest components of total soluble SS activity, respectively, in developing japonica rice (Oryza sativa L.) endosperm. To elucidate the distinct and overlapping functions of these enzymes, double mutants were created by crossing the ss1 null mutant with the ss3a null mutant. In the F(2) generation, two opaque seed types were found to have either the ss1ss1/SS3ass3a or the SS1ss1/ss3ass3a genotype. Phenotypic analyses revealed lower SS activity in the endosperm of these lines than in those of the parent mutant lines since these seeds had different copies of SSI and SSIIIa genes in a heterozygous state. The endosperm of the two types of opaque seeds contained the unique starch with modified fine structure, round-shaped starch granules, high amylose content, and specific physicochemical properties. The seed weight was ～90% of that of the wild type. The amount of granule-bound starch synthase I (GBSSI) and the activity of ADP-glucose pyrophosphorylase (AGPase) were higher than in the wild type and parent mutant lines. The double-recessive homozygous mutant prepared from both ss1 and ss3a null mutants was considered sterile, while the mutant produced by the leaky ss1 mutant×ss3a null mutant cross was fertile. This present study strongly suggests that at least SSI or SSIIIa is required for starch biosynthesis in rice endosperm.     

Conformational transformation and selection of synthetic prion strains

Prion protein is capable of folding into multiple self-replicating prion strains that produce phenotypically distinct neurological disorders. Although prion strains often breed true upon passage, they can also transform or “mutate” despite being devoid of nucleic acids. To dissect the mechanism of prion strain transformation, we studied the physicochemical evolution of a mouse synthetic prion (MoSP) strain, MoSP1, after repeated passage in mice and cultured cells. We show that MoSP1 gradually adopted shorter incubation times and lower conformational stabilities. These changes were accompanied by structural transformation, as indicated by a shift in the molecular mass of the protease-resistant core of MoSP1 from approximately 19 kDa [MoSP1(2)] to 21 kDa [MoSP1(1)]. We show that MoSP1(1) and MoSP1(2) can breed with fidelity when cloned in cells; however, when present as a mixture, MoSP1(1) preferentially proliferated, leading to the disappearance of MoSP1(2). In culture, the rate of this transformation process can be influenced by the composition of the culture media and the presence of polyamidoamines. Our findings demonstrate that prions can exist as a conformationally diverse population of strains, each capable of replicating with high fidelity. Rare conformational conversion, followed by competitive selection among the resulting pool of conformers, provides a mechanism for the adaptation of the prion population to its host environment.     

Alkoxy-auxins are selective inhibitors of auxin transport mediated by PIN, ABCB, and AUX1 transporters

Polar auxin movement is a primary regulator of programmed and plastic plant development. Auxin transport is highly regulated at the cellular level and is mediated by coordinated transport activity of plasma membrane-localized PIN, ABCB, and AUX1/LAX transporters. The activity of these transporters has been extensively analyzed using a combination of pharmacological inhibitors, synthetic auxins, and knock-out mutants in Arabidopsis. However, efforts to analyze auxin-dependent growth in other species that are less tractable to genetic manipulation require more selective inhibitors than are currently available. In this report, we characterize the inhibitory activity of 5-alkoxy derivatives of indole 3-acetic acid and 7-alkoxy derivatives of naphthalene 1-acetic acid, finding that the hexyloxy and benzyloxy derivatives act as potent inhibitors of auxin action in plants. These alkoxy-auxin analogs inhibit polar auxin transport and tropic responses associated with asymmetric auxin distribution in Arabidopsis and maize. The alkoxy-auxin analogs inhibit auxin transport mediated by AUX1, PIN, and ABCB proteins expressed in yeast. However, these analogs did not inhibit or activate SCF(TIR1) auxin signaling and had no effect on the subcellular trafficking of PIN proteins. Together these results indicate that alkoxy-auxins are inactive auxin analogs for auxin signaling, but are recognized by PIN, ABCB, and AUX1 auxin transport proteins. Alkoxy-auxins are powerful new tools for analyses of auxin-dependent development.     

Site-1 protease is essential to growth plate maintenance and is a critical regulator of chondrocyte hypertrophic differentiation in postnatal mice

Site-1 protease (S1P) is a proprotein convertase with essential functions in lipid homeostasis and unfolded protein response pathways. We previously studied a mouse model of cartilage-specific knock-out of S1P in chondroprogenitor cells. These mice exhibited a defective cartilage matrix devoid of type II collagen protein (Col II) and displayed chondrodysplasia with no endochondral bone formation even though the molecular program for endochondral bone development appeared intact. To gain insights into S1P function, we generated and studied a mouse model in which S1P is ablated in postnatal chondrocytes. Postnatal ablation of S1P results in chondrodysplasia. However, unlike early embryonic ablations, the growth plates of these mice exhibit a lack of Ihh, PTHrP-R, and Col10 expression indicating a loss of chondrocyte hypertrophic differentiation and thus disruption of the molecular program required for endochondral bone development. S1P ablation results in rapid growth plate disruption due to intracellular Col II entrapment concomitant with loss of chondrocyte hypertrophy suggesting that these two processes are related. Entrapment of Col II in the chondrocytes of the prospective secondary ossification center precludes its development. Trabecular bone formation is dramatically diminished in the primary spongiosa and is eventually lost. The primary growth plate is eradicated by apoptosis but is gradually replaced by a fully functional new growth plate from progenitor stem cells capable of supporting new bone growth. Our study thus demonstrates that S1P has fundamental roles in the preservation of postnatal growth plate through chondrocyte differentiation and Col II deposition and functions to couple growth plate maturation to trabecular bone development in growing mice.