Novel lipoxygenase inhibitors, tetrapetalone B, C, and D from Streptomyces sp

Three novel lipoxygenase inhibitors, tetrapetalone B (2, C(28)H(35)NO(9)), C (3, C(26)H(34)NO(8)), and D (4, C(28)H(36)NO(10)), were isolated from a culture broth of Streptomyces sp. USF-4727 that produced a lipoxygenase inhibitor tetrapetalone A (1) simultaneously. Each chemical structure was revealed by spectroscopic evidence, this suggests that these three compounds are structurally related to 1. They had a tetracyclic skeleton and a beta-D-rhodinosyl moiety. Tetrapetalone B, C, and D inhibited soybean lipoxygenase with IC(50): 320, 360, and 340 microM respectively.     

Pterygodermatites nycticebi (Nematoda: Rictulariidae): accidental detection of encapsulated third-stage larvae in the tissue of a white-fronted marmoset

Twin, white-fronted marmosets (Callithrix geoffroyi) born and raised in a zoo in Japan died at 7 mo of age. Several encapsulated nematode larvae were detected in the intestinal wall, as well as a few in the mesenteric lymph nodes of 1 of the twins. In the other marmoset, no encapsulated nematode larva was detected in the organs, but many adult Pterygodermatites nycticebi were found in the intestinal lumen. In the past 5 yr, 5 primates kept in the same zoo, i.e., 1 squirrel monkey (Saimiri sciureus), 2 Pygmy marmosets (Cebuella pygmaea), 1 Senegal galago (Galago senegalensis), and 1 cotton-top tamarin (Saguinus oedipus), died from heavy infestation with the same nematode. A few migrating larvae of the rictulariid were also identified histologically in the intestinal wall and liver of the cotton-top tamarin. Although no other primate currently held in the same zoo was infected with the rictulariid, German cockroaches (Blattella germanica) collected with traps near marmoset cages had encapsulated P. nycticebi larvae, indicating latent perpetuation of the life cycle of this rictulariid species in the zoo premises. Our results indicated that encapsulation or migration of third-stage larvae of P. nycticebi might occur accidentally in the organs of callithrichid primates.     

Construction of reporter yeasts for mouse aryl hydrocarbon receptor ligand activity

Aryl hydrocarbons such as dioxins, polychlorinated biphenyls and polyaromatic hydrocarbons bind to the cellular aryl hydrocarbon receptor (AhR) in the initial step of their metabolism. The activation of intracellular signaling subsequent to the AhR binding is highly correlated with the toxicity and carcinogenicity of these chemicals. We produced Saccharomyces cerevisiae coexpressing mouse AhR and aryl hydrocarbon receptor nuclear translocator (Arnt) protein in accordance with Miller III's method for constructing yeasts with human Ahr and Arnt [Toxicol. Appl. Pharmacol. 160 (1998) 297]. Ligand treatment induced a dose-dependent increase in beta-galactosidase activity from a reporter plasmid in the yeast. Then, we compared activities of several ligands in yeast having the mouse Ahr/Arnt genes with those in yeast having the human genes, both of which have the same genetic background. There was no significant difference in the EC50 values of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo[a]pyrene, 3-methylcholanthrene and beta-naphthoflavone between the mouse and human genes. However, indirubin, which was recently found in human urine as a potent AhR ligand [J. Biol. Chem. 276 (2001) 31475], had a 35-140 times higher EC50 value in the yeast with human genes than mouse genes. This difference might reflect species-specificity between mouse and human AhR/Arnt.     

Molecular, biochemical and immunological analyses of canine pancreatic DNase I

The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.     

Role of TGF-beta in the retinoic acid-induced inhibition of proliferation and melanin synthesis in chick retinal pigment epithelial cells in vitro

The purpose of this study was to investigate the effects of all-trans retinoic acid (RA) on the induction of transforming growth factor-beta (TGF-beta) that is concerned with the proliferation and melanin synthesis of chick retinal pigment epithelial (RPE) cells in vitro. Chick RPE cells were cultured in the presence or absence of RA and anti-TGF-beta antibody for 7 days. The effects of RA and pan-specific TGF-beta antibody on RPE cell proliferation were assessed by counting the number of cells, and their effects on melanin synthesis were evaluated by measuring the melanin content of the cells. TGF-beta activity in the culture supernatant of RPE cells was measured using CCL-64 cells. RA significantly inhibited RPE cell proliferation and increased melanin synthesis. The addition of pan-specific TGF-beta antibody to the culture blocked the inhibition of RPE cell proliferation and the increased melanin synthesis. RA induced TGF-beta production in the culture supernatant of RPE cells. These findings indicate that RA regulates the proliferation and melanin synthesis of RPE cells via induction of TGF-beta.     

Identification of two distinct structural motifs that, when added to the C-terminal tail of the rat LH receptor, redirect the internalized hormone-receptor complex from a degradation to a recycling pathway

We show that most of the internalized rat LH receptor is routed to a lysosomal degradation pathway whereas a substantial portion of the human LH receptor is routed to a recycling pathway. Chimeras of these two receptors identified a linear amino acid sequence (GTALL) present near the C terminus of the human LH receptor that, when grafted onto the rat LH receptor, redirects most of the rat LH receptor to a recycling pathway. Removal of the GTALL sequence from the human LH receptor failed to affect its routing, however. The GTALL sequence shows homology with the C-terminal tetrapeptide (DSLL) of the beta2-adrenergic receptor, a motif that has been reported to mediate the recycling of the internalized beta2-adrenergic receptor by binding to ezrin-radixin-moesin-binding phosphoprotein-50. Addition of the DSLL tetrapeptide to the C terminus of the rat LH receptor also redirects most of the internalized rat LH receptor to a recycling pathway but, like the recycling of the human LH receptor, this rerouting is not mediated by ezrin-radixin-moesin-binding phosphoprotein-50. We conclude that most of the internalized rat LH receptor is degraded because its C-terminal tail lacks motifs that promote recycling and that two distinct, but homologous, motifs (DSLL at the C terminus or GTALL near the C terminus) can reroute the internalized rat LH receptor to a recycling pathway that is independent of ezrin-radixin-moesin-binding phosphoprotein-50.     

Involvement of the telomeric protein Pin2/TRF1 in the regulation of the mitotic spindle

Pin2/TRF1 was independently identified as a telomeric DNA-binding protein (TRF1) that regulates telomere length, and as a protein (Pin2) that can bind the mitotic kinase NIMA and suppress its lethal phenotype. We have previously demonstrated that Pin2/TRF1 levels are cell cycle-regulated and its overexpression induces mitotic arrest and then apoptosis. This Pin2/TRF1 activity can be potentiated by microtubule-disrupting agents, but suppressed by phosphorylation of Pin2/TRF1 by ATM; this negative regulation is critical in mediating for many, but not all, ATM-dependent phenotypes. Interestingly, Pin2/TRF1 specifically localizes to mitotic spindles in mitotic cells and affects the microtubule polymerization in vitro. These results suggest a role of Pin2/TRF1 in mitosis. However, nothing is known about whether Pin2/TRF1 affects the spindle function in mitotic progression. Here we characterized a new Pin2/TRF1-interacting protein, EB1, that was originally identified in our yeast two-hybrid screen. Pin2/TRF1 bound EB1 both in vitro and in vivo and they also co-localize at the mitotic spindle in cells. Furthermore, EB1 inhibits the ability of Pin2/TRF1 to promote microtubule polymerization in vitro. Given that EB1 is a microtubule plus end-binding protein, these results further confirm a specific interaction between Pin2/TRF1 and the mitotic spindle. More importantly, we have shown that inhibition of Pin2/TRF1 in ataxia-telangiectasia cells is able to fully restore their mitotic spindle defect in response to microtubule disruption, demonstrating for the first time a functional involvement of Pin2/TRF1 in mitotic spindle regulation.     

A novel class II-binding motif selects peptides that mediate organ-specific autoimmune disease in SWXJ,SJL/J,and SWR/J mice

Idiopathic dilated cardiomyopathy (DCM) is responsible for approximately 25% of all cases of congestive heart failure. We have recently shown that immunization of autoimmune-susceptible SWXJ mice with whole cardiac myosin leads to T cell-mediated experimental autoimmune myocarditis (EAMC) and DCM. We have now identified two disease-inducing peptides from cardiac alpha-myosin heavy chain (CAMHC). Our approach involved the use of a novel MHC class II-binding motif contained in several peptides known to be immunogenic in SWXJ (H-2(q,s)) mice or in the parental SJL/J (H-2(s)) or SWR/J (H-2(q)) mouse strains. Two of four CAMHC peptides containing the -KXXS- peptide motif were found to be immunogenic. Immunization of SWXJ or parental SJL/J and SWR/J mice with CAMHC peptides palpha406-425 or palpha1631-1650 resulted in EAMC and DCM, characterized by inflammation, fibrosis, and decompensated right-sided ventricular dilatation. Despite mediating high incidences of severe disease, both peptides were found to be cryptic determinants, thereby providing further evidence for the importance and perhaps predominance of self crypticity in autoimmunity. Both peptides showed dual parental I-A(q) and I-A(s) restriction and mediated passive transfer of disease with activated CD4(+) T cells. An intact motif was necessary for antigenicity because loss of activity occurred in peptides containing nonconservative substitutions at the motif's terminal lysine and serine residues. Our studies provide a new model for EAMC and DCM in strains of mice widely used in autoimmune studies. Moreover, the -KXXS- motif may be particularly useful in implicating previously overlooked proteins as autoimmune targets and in facilitating the development of new organ-specific autoimmune mouse models for human diseases.