Elucidation of the role of COX-2 in liver fibrogenesis using transgenic mice

Hepatic COX-2 overexpression is sufficient to induce hepatitis, but its role on liver fibrosis remains unknown. We aim to elucidate possible biological effects of COX-2 in liver fibrosis using both gain-of-function and loss-of-function mouse models. COX-2 transgenic (TG) mice that specifically overexpress the human COX-2 cDNA in the liver, knockout (KO), and wild type (WT) mice were studied in two different murine fibrosis models induced by carbon tetrachloride (CCl₄) injection or methionine and choline-deficient (MCD) diet. Liver injury was assessed by serum ALT and bilirubin levels and histological examination. Hepatic collagen content was determined by picrosirius red stain morphometry assay and quantitation of hydroxyproline. Hepatic stellate cell (HSC) activation was determined by immunohistochemical analysis of α-smooth muscle actin (α-SMA). mRNA expression of fibrogenic genes was assayed by real-time quantitative PCR. COX-2 protein was overexpressed in the liver of TG mice compared with WT littermates. CCl₄ or MCD-induced liver fibrotic injury was equally severe in TG and WT mice, as demonstrated by similar elevated levels of hepatic collagen contents. Enhanced COX-2 expression in TG liver did not affect HSC activation and fibrogenic gene expression upon CCl₄ or MCD treatment. Importantly, CCl₄-treated KO mice did not show significant difference in liver fibrotic damage and fibrogenic gene expression compared with the WT counterparts. This is the first report on the effect of COX-2 in liver fibrosis based on genetic mouse models. The results suggest that COX-2 does not appear to mediate the development of liver fibrosis.     

Reactive oxygen species up-regulate CD11b in microglia via nitric oxide: Implications for neurodegenerative diseases

Microglial activation is considered as a hallmark of several neurodegenerative disorders. During microglial activation, the expression of CD11b, the beta-integrin marker of microglia, is increased. However, the molecular mechanism behind increased microglial CD11b expression is poorly understood. The present study was undertaken to explore the role of reactive oxygen species (ROS) in the expression of CD11b in microglial cells. Bacterial lipopolysaccharide (LPS) stimulated the expression of CD11b in mouse BV-2 microglial cells and primary microglia, the effect that was blocked by antioxidants such as N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). Furthermore, comicroinjection of either NAC or PDTC with LPS was also able to suppress LPS-stimulated expression of CD11b in striatum in vivo. Similarly, other neurotoxic molecules, such as interleukin-1beta (IL-1beta), IL-12 p40(2), fibrillar amyloid-beta (Abeta) peptides, HIV-1 gp120, and double-stranded RNA (poly(IC)), also stimulated the expression of CD11b in microglia through the involvement of ROS. Complete inhibition of LPS-stimulated expression of CD11b by catalase, induction of CD11b expression by H2O2 alone, and inhibition of superoxide-stimulated CD11b expression by catalase suggest that H2O2, but not superoxide, is in fact involved in the expression of CD11b. Interestingly, we also demonstrate that ROS stimulated the expression of CD11b after the induction of nitric oxide (NO) production and failed to stimulate CD11b when NO production was inhibited by either 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) or L-N6-(1-iminoethyl)-L-lysine (L-NIL). Taken together, these studies suggest that the up-regulation of CD11b in microglia is redox sensitive and that ROS up-regulates CD11b via NO.     

Schisandrin B protects against menadione-induced hepatotoxicity by enhancing DT-diaphorase activity

Pretreating mice with schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, at a daily dose of 1 mmol/kg for 3 days protected against menadione-induced hepatic oxidative damage in mice, as evidenced by decreases in plasma alanine aminotransferase activity (78%) and hepatic malondialdehyde level (70%), when compared with the menadione intoxicated control. In order to define the biochemical mechanism involved in the hepatoprotection afforded by Sch B pretreatment, we examined the activity of DT-diaphorase (DTD) in hepatocytes isolated from Sch B pretreated rats. Hepatocytes isolated from Sch B pretreated (a daily dose of 1 mmol/kg for 3 days) rats showed a significant increase (25%) in DTD activity. The increase in DTD activity was associated with the enhanced rate of menadione elimination in the hepatocyte culture. The ensemble of results suggests that the ability of Sch B pretreatment to enhance hepatocellular DTD activity may at least in part be attributed to the protection against menadione hepatotoxicity.     

Antineuroinflammatory effect of NF-kappaB essential modifier-binding domain peptides in the adoptive transfer model of experimental allergic encephalomyelitis

It has been shown that peptides corresponding to the NF-kappaB essential modifier-binding domain (NBD) of IkappaB kinase alpha or IkappaB kinase beta specifically inhibit the induction of NF-kappaB activation without inhibiting the basal NF-kappaB activity. The present study demonstrates the effectiveness of NBD peptides in inhibiting the disease process in adoptively transferred experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis. Clinical symptoms of EAE were much lower in mice receiving wild-type (wt)NBD peptides compared with those receiving mutated (m)NBD peptides. Histological and immunocytochemical analysis showed that wtNBD peptides inhibited EAE-induced spinal cord mononuclear cell invasion and normalized p65 (the RelA subunit of NF-kappaB) expression within the spinal cord. Analysis of lymph node cells isolated from donor and recipient mice showed that wtNBD peptides but not mNBD peptides were able to shift the immune response from a Th1 to a Th2 profile. Consistently, wtNBD peptides but not mNBD peptides inhibited the encephalitogenicity of myelin basic protein-specific T cells. Furthermore, i.p. injection of wtNBD peptides but not mNBD peptides was also able to reduce LPS- and IFN-gamma-induced expression of inducible NO synthase, IL-1beta, and TNF-alpha in vivo in the cerebellum. Taken together, our results support the conclusion that NBD peptides are antineuroinflammatory, and that NBD peptides may have therapeutic effect in neuroinflammatory disorders such as multiple sclerosis.     

Female mate preference based on male orange spot patterns in the feral guppy <Emphasis Type="Italic">Poecilia reticulata</Emphasis> in Japan

It is known that females prefer males with larger and/or brighter orange spots in many populations of the guppy Poecilia reticulata. However, female preference for male orange spots varies among populations and changes within several years when they are introduced into new habitats with different environment. Guppies were introduced into Okinawa, Japan, more than 20 years ago and were subjected to natural and sexual selection for a long period. The female preference for orange spot patterns of males was examined by the dichotomous choice experiment for a feral guppy population of the Hiji River, Okinawa. We chose full-sibling males as a pair of stimulus males that were simultaneously presented to a test female, because sibling males should resemble each other. To create different orange spot patterns between stimulus males, one male of the stimulus male pair was fed carotenoid-supplement food such as algae and another male was fed low-carotenoid food. High-carotenoid-treatment males showed not only brighter coloration of orange spots but also larger spots than other males as a result of this dietary-manipulation. In the dichotomous choice experiment, females preferred the high-carotenoid-treatment males. In addition, logistic regression analysis clarified that brighter coloration of male orange spots was the most important factor for female mate preference. This finding suggests indirect benefits of female preference for male orange spot patterns if the male foraging ability for algae were heritable.     

An epididymis-specific beta-defensin is important for the initiation of sperm maturation

Although the role of the epididymis, a male accessory sex organ, in sperm maturation has been established for nearly four decades, the maturation process itself has not been linked to a specific molecule of epididymal origin. Here we show that Bin1b, a rat epididymis-specific beta-defensin with antimicrobial activity, can bind to the sperm head in different regions of the epididymis with varied binding patterns. In addition, Bin1b-expressing cells, either of epididymal origin or from a Bin1b-transfected cell line, can induce progressive sperm motility in immotile immature sperm. This induction of motility is mediated by the Bin1b-induced uptake of Ca(2+), a mechanism that has a less prominent role in maintaining motility in mature sperm. In vivo antisense experiments show that suppressed expression of Bin1b results in reduced binding of Bin1b to caput sperm and in considerable attenuation of sperm motility and progressive movement. Thus, beta-defensin is important for the acquisition of sperm motility and the initiation of sperm maturation.     

The role of inducible nitric oxide synthase in gamete interaction and fertilization: a comparative study on knockout mice of three NOS isoforms

Nitric oxide (NO), which is produced from l-arginine by three isoforms of NO synthase (NOS), has been implicated in reproductive functions. However, the specific role of NOS isoforms in gamete function and fertilization is not clear. Three types of NOS knockout mice were super ovulated and fertilized in vitro and in vivo. The sperm count and motility, in vivo and in vitro fertilization rate as indicated by two-cell embryos and blastocyst rate were examined. The sperm count and motility from all three knockout mice were not significantly different from that of the wild type. Inducible NOS (iNOS) knockout mice were found to have the largest number of two-cell embryos/mouse collected after fertilization in vivo (P<0.01), but the rate of blastocyst formation from two-cell embryos in vitro was similar for all three knockouts. The rate of in vitro fertilization using either iNOS-deficient sperm or oocytes, but not those deficient in the other two NOS isoforms, was significantly elevated when compared to that in the wild type (P<0.001). While all three types of NOS do not seem to play a significant role in pre-ejaculated sperm function, iNOS may play an inhibitory role in sperm and oocyte functions affecting the process of fertilization and early embryo development.     

The mutated subsequence problem and locating conserved genes

MOTIVATION: For the purpose of locating conserved genes in a whole genome scale, this paper proposes a new structural optimization problem called the Mutated Subsequence Problem, which gives consideration to possible mutations between two species (in the form of reversals and transpositions) when comparing the genomes. RESULTS: A practical algorithm called mutated subsequence algorithm (MSS) is devised to solve this optimization problem, and it has been evaluated using different pairs of human and mouse chromosomes, and different pairs of virus genomes of Baculoviridae. MSS is found to be effective and efficient; in particular, MSS can reveal >90% of the conserved genes of human and mouse that have been reported in the literature. When compared with existing softwares MUMmer and MaxMinCluster, MSS uncovers 14 and 7% more genes on average, respectively. Furthermore, this paper shows a hybrid approach to integrate MUMmer or MaxMinCluster with MSS, which has better performance and reliability.     

Bak Foong Pills stimulate anion secretion across normal and cystic fibrosis pancreatic duct epithelia

The present study examined the effect of Bak Foong Pills (BFP), an over-the-counter traditional Chinese medicine (China registration no. Z980035), on anion secretion and the underlying signaling pathways in normal and cystic fibrosis pancreatic duct cell lines, CAPAN-1 and CFPAC-1, respectively, using the short-circuit current technique. Apical addition of BFP ethanol extract (600 microg/ml) induced a fast transient I(SC) peak that was followed by a slower but more sustained increase in I(SC) in CAPAN-1 cells. However, the response to BFP in CFPAC-1 was predominantly the first transient peak. Apical addition of DIDS (200 microM) inhibited the first peak by more than 60% in both cell lines without significantly affecting the second I(SC) rise. More than 85% of the BFP-induced first transient in both cell lines was inhibited when extra and intracellular Ca(2+) was chelated or emptied by pre-treatment with BAPTA (100 microM) and thapsigargin (10 microM), respectively. Acute addition of PMA (1 microM), a PKC activator, blocked more than 95% of the BFP-induced first peak in both cell lines, consistent with previously reported PKC modulation of Ca(2+)-dependent pancreatic anion secretion. The BFP-induced second I(SC) rise in CAPAN-1 could be inhibited by 73.6% and 71.13% by pretreatment of the cells with MDL-12330A (20 microM), an adenylate cyclase inhibitor and Rp-cAMP (200 microM), a cyclic AMP antagonist, respectively. However, less than 25% of the I(SC) was inhibited by combined treatment with BAPTA and thapsigargin. The second rise was also completely blocked by DPC (2mM) or Glibenclamide (1mM). The results indicate that BFP ethanol extract stimulates pancreatic duct anion secretion in normal and CF cells via different signaling pathways involving both Ca(2+) and cAMP.