RNAi-mediated knock-down of arylamine N-acetyltransferase-1 expression induces E-cadherin up-regulation and cell-cell contact growth inhibition

Arylamine N-acetyltransferase-1 (NAT1) is an enzyme that catalyzes the biotransformation of arylamine and hydrazine substrates. It also has a role in the catabolism of the folate metabolite p-aminobenzoyl glutamate. Recent bioinformatics studies have correlated NAT1 expression with various cancer subtypes. However, a direct role for NAT1 in cell biology has not been established. In this study, we have knocked down NAT1 in the colon adenocarcinoma cell-line HT-29 and found a marked change in cell morphology that was accompanied by an increase in cell-cell contact growth inhibition and a loss of cell viability at confluence. NAT1 knock-down also led to attenuation in anchorage independent growth in soft agar. Loss of NAT1 led to the up-regulation of E-cadherin mRNA and protein levels. This change in E-cadherin was not attributed to RNAi off-target effects and was also observed in the prostate cancer cell-line 22Rv1. In vivo, NAT1 knock-down cells grew with a longer doubling time compared to cells stably transfected with a scrambled RNAi or to parental HT-29 cells. This study has shown that NAT1 affects cell growth and morphology. In addition, it suggests that NAT1 may be a novel drug target for cancer therapeutics.     

Defensive burying: Effects of diazepam and oxprenolol measured in extinction

When a rat is shocked via a prod in a chamber with sawdust on the floor it will typically push the flooring material with snout and forepaws towards and over the prod. We administered diazepam (.5 and 1.0 mg/kg) and oxprenolol (10 and 20 mg/kg) the day following shock exposure, and observed the complete suppression of burying by diazepam, and some suppression with oxprenolol. These effects are independent of interference with initial association of shock and prod, and of changes in general activity.     

A field study into the appropriateness of transcutaneous ultrasonography in the diagnoses of uterine disorders in reproductively failed pigs

This study was conducted to define the characteristics of the uterus of reproductively failed pigs by transcutaneous ultrasonography (SONO) in order to investigate the appropriateness of SONO to diagnose presumptive uterine disorders. Zearalenone (ZEA) is known to affect uterine function and causes endometrial liquid accumulation and was also determined. In 33 sows and 14 gilts, of unknown reproductive stages and culled for failing to conceive, the uterus was scanned transcutaneously and the females slaughtered on the same day or the day after scanning. Parameters determined by SONO were uterine echotexture (UET; graded 1 for homogeneous to 4 for highly heterogeneous), uterine size (US; expressed as the mean sectional area of two to three cross-sections of the uterine horns given in cm2) and intrauterine content. Post mortem, the ovarian structures were assessed and females grouped accordingly into those in estrus (n=2), early diestrus (n=14), diestrus (n=15), late diestrus (n=1), anestrus (n=10) and having polycystic ovarian degeneration (n=5). The uterine weight (UW) was recorded and uterine specimens microscopically evaluated for an endometrial oedema (EO; grades 1 for none, to 4, if an oedema was clearly evident) and for immune cells to assess endometritis. Total ZEA was analysed in bile and females with >or=50 ng/ml judged as positive. The uterus could be examined in all animals. UET, US, UW and EO was found to be different between groups, and a positive correlation (P<0.001) established for US and UET (r=0.71), US and EO (r=0.51), UET and EO (r=0.57), US and UW (UW=357.6 x US(0.801); r=0.88). One female had intrauterine fluid and an acute-chronic endometritis diagnosed. Almost all females had a chronic endometritis and a majority found ZEA positive. No differences were observed between groups and a relationship between ZEA or chronic endometritis and UET, US, UW and EO were not established. In conclusion, transcutaneous SONO is appropriate to examine the uterus in reproductively failed pigs on farms, and the estimation of UET and US gives information on EO and UW. Intrauterine fluid is indicative for a severe uterine inflammation. Since groups differed in UET and US, but were equally ZEA positive and the uteri chronically inflamed, an UET and US specifically associated with ZEA or chronic endometritis is questioned.     

Structural and functional modeling of human lysozyme reveals a unique nonapeptide, HL9, with anti-HIV activity

We previously reported that lysozyme accounts for anti-HIV activity associated with the beta-core fraction of human chorionic gonadotropin [Lee-Huang, S., Huang, P. L., Sun, Y., Kung, H. F., Blithe, D. L. & Chen, H. C. (1999) Proc Natl Acad Sci U S A 96, 2678-81]. To define the structural and sequence requirements for anti-HIV activity, we carried out peptide fragmentation and activity mapping of human lysozyme. We identified two peptides that consist of 18 and 9 amino acids of human lysozyme (HL18 and HL9), corresponding to residues 98-115 and 107-115. HL18 and HL9 are potent inhibitors of HIV-1 infection and replication with EC(50)s of 50 to 55 nM, comparable to intact lysozyme. Scrambling the sequence or substitution of key arginine or tryptophan residues results in loss of antiviral activity. HL9, with the sequence RAWVAWRNR, is the smallest peptide we identified with full anti-HIV activity. It forms a pocket with its basic residues on the surface of the molecule. HL9 exists as an alpha-helix in native human lysozyme, in a region of the protein distinct from the muramidase catalytic site. Monte Carlo peptide folding energy minimizing simulation modeling and CD studies indicate that helical propensity does not correlate with antiviral activity. HL9 blocks HIV-1 viral entrance and replication, and modulates gene expression of HIV-infected cells, affecting pathways involved in survival, stress, TGFbeta, p53, NFkappaB, protein kinase C and hedgehog signaling.     

Accumulation of glycosphingolipids in Niemann-Pick C disease disrupts endosomal transport

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus but are mistargeted to lysosomes in sphingolipid storage disorders. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Niemann-Pick type C (NPC) disease is a disorder of intracellular transport where glycosphingolipids (GSLs) and cholesterol accumulate in endosomal compartments. The mechanisms of altered intracellular trafficking are not known but may involve the mistargeting and disrupted function of proteins associated with GSL membrane microdomains. Membrane microdomains were isolated by Triton X-100 and sucrose density gradient ultracentrifugation. High pressure liquid chromatography and mass spectrometric analysis of NPC1(-/-) mouse brain revealed large increases in GSL. Sphingosine was also found to be a component of membrane microdomains, and in NPC liver and spleen, large increases in cholesterol and sphingosine were found. GSL and cholesterol levels were increased in mutant NPC1-null Chinese hamster ovary cells as well as U18666A and progesterone induced NPC cell culture models. However, inhibition of GSL synthesis in NPC cells with N-butyldeoxygalactonojirimycin led to marked decreases in GSL but only small decreases in cholesterol levels. Both annexin 2 and 6, membrane-associated proteins that are important in endocytic trafficking, show distorted distributions in NPC cells. Altered BODIPY lactosylceramide targeting, decreased endocytic uptake of a fluid phase marker, and mistargeting of annexin 2 (phenotypes associated with NPC) are reversed by inhibition of GSL synthesis. It is suggested that accumulating GSL is part of a mislocalized membrane microdomain and is responsible for the deficit in endocytic trafficking found in NPC disease.     

Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: identification and characterisation of an elongation factor 1B protein with glutathione transferase activity

Aspergillus fumigatus is a recognised human pathogen, especially in immunocompromised individuals. The availability of the annotated A. fumigatus genome sequence will significantly accelerate our understanding of this organism. However, limited information is available with respect to the A. fumigatus proteome. Here, both a direct proteomic approach (2D-PAGE and MALDI-MS) and a sub-proteomic strategy involving initial glutathione affinity chromatography have been deployed to identify 54 proteins from A. fumigatus primarily involved in energy metabolism and protein biosynthesis. Furthermore, two novel eukaryotic elongation factor proteins (eEF1Bgamma), termed ElfA and B have been identified and phylogenetically confirmed to belong to the eEF1Bgamma class of GST-like proteins. One of these proteins (ElfA) has been purified to homogeneity, identified as a monomeric enzyme (molecular mass=20 kDa; pI=5.9 and 6.5), and found to exhibit glutathione transferase activity specific activities (mean+/-standard deviation, n=3) of 3.13+/-0.27 and 3.43+/-1.0 micromol/min/mg, using CDNB and ethacrynic acid, respectively. Overall, these data highlight the importance of new approaches to dissect the proteome of, and elucidate novel functions within, A. fumigatus.