Evidence that exogenous substances can be phagocytized by alveolar epithelial cells and transported into blood capillaries

Since the ability of alveolar epithelial cells to ingest inhaled fine particles has not been characterized in detail, the present study seeks to evaluate this physiological activity. We used a 0.2% suspension of intact or lecithin-coated polystyrene latex beads (240 nm in diameter). A 5-ml suspension of intact or lecithin-coated latex beads was intratracheally administered to rats using a compressor nebulizer. Thereafter, the lungs were perfused intratracheally with glutaraldehyde solution and cut into small pieces. The samples were postfixed with osmium tetroxide, embedded in epoxy resin and examined under an electron microscope. Both lecithin-coated and uncoated beads were incorporated into alveolar macrophages. Some of the ingested beads in the alveolar macrophages were sequestered within lysosomes. Types I and II alveolar epithelial cells selectively incorporated only lecithin-coated beads, which were also observed within the cytoplasm of monocytes in the capillary lumen. These findings suggest that alveolar epithelial cells can incorporate exogenous particles, which are then transferred from the alveoli to intravascular spaces by transcytosis.     

Cellular events involved in butyric acid-induced T cell apoptosis

We have previously demonstrated that butyric acid induces cytotoxicity and apoptosis of murine thymocytes, splenic T cells, and human Jurkat T cells. Therefore, to determine the apoptotic signaling pathway induced by butyric acid, we investigated the contribution of reactive oxygen species (ROS), mitochondria, ceramide, and mitogen-activated protein kinases in butyric acid-induced human Jurkat cell apoptosis. After exposure of cells to butyric acid, a pronounced accumulation of ROS was seen. Pretreatment of cells with the antioxidant N-acetyl-cysteine or 3-aminobenzamide attenuated butyric acid-induced apoptosis through a reduction of ROS generation. Cytochrome c, apoptosis-inducing factor, and second mitochondria-derived activator of caspases protein release from mitochondria into the cytosol were detected shortly after butyric acid treatment. Exposure of cells to butyric acid resulted in an increase in cellular ceramide in a time-dependent fashion. In addition, butyric acid-induced apoptosis was inhibited by DL-threo-dihidrosphingosine, a potent inhibitor of sphingosine kinase. Using anti-extracellular signal-regulated kinase (ERK), anti-c-Jun N-terminal kinase (JNK), and anti-p38 phosphospecific Abs, we showed a decrease in ERK, but not in JNK and p38 phosphorylation after treatment of cells with butyric acid. Pretreatment of cells with the JNK inhibitor SP600125 attenuated the effect of butyric acid on apoptosis, whereas no effect was seen with the p38 inhibitor SB202190 or the ERK inhibitor PD98059. Taken together, our results indicate that butyric acid-induced T cell apoptosis is mediated by ceramide production, ROS synthesis in mitochondria, and JNK activation in the mitogen-activated protein kinase cascade. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

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.     

Characterization of cluster N5 as a fast-relaxing [4Fe-4S] cluster in the Nqo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans

The NADH-quinone oxidoreductase from Paracoccus denitrificans consists of 14 subunits (Nqo1-14) and contains one FMN and eight iron-sulfur clusters. The Nqo3 subunit possesses fully conserved 11 Cys and 1 His in its N-terminal region and is considered to harbor three iron-sulfur clusters; however, only one binuclear (N1b) and one tetranuclear (N4) were previously identified. In this study, the Nqo3 subunit containing 1x[2Fe-2S] and 2x[4Fe-4S] clusters was expressed in Escherichia coli. The second [4Fe-4S](1+) cluster is detected by EPR spectroscopy below 6 K, exhibiting very fast spin relaxation. The resolved EPR spectrum of this cluster is broad and nearly axial. The subunit exhibits an absorption-type EPR signal around g approximately 5 region below 6 K, most likely arising from an S = 3/2 ground state of the fast-relaxing [4Fe-4S](1+) species. The substitution of the conserved His(106) with Cys specifically affected the fast-relaxing [4Fe-4S](1+) cluster, suggesting that this cluster is coordinated by His(106). In the cholate-treated NDH-1-enriched P. denitrificans membranes, we observed EPR signals arising from a [4Fe-4S] cluster below 6 K, exhibiting properties similar to those of cluster N5 detected in other complex I/NDH-1 and of the fast-relaxing [4Fe-4S](1+) cluster in the expressed Nqo3 subunit. Hence, we propose that the His-coordinated [4Fe-4S] cluster corresponds to cluster N5.     

Levels and behavior of 2-phenylbenzotoriazole-type mutagens in the effluent of a sewage treatment plant

We previously reported on the isolation and structural determination of five 2-phenylbenzotriazole (PBTA)-type mutagens (PBTA-1, PBTA-2, PBTA-3, PBTA-4 and PBTA-6) in blue rayon/cotton adsorbed substances collected from surface waters at sites located downstream of sewage treatment plants. We also noted that PBTA-1 and PBTA-2 were discharged from sewage treatment plants and subsequently diluted or decomposed while moving down the Yodo River system. However, it has not been investigated whether they are commonly discharged from sewage treatment plants into rivers. The main purpose of this study was to make a comprehensive survey of levels and behavior of PBTA-type mutagens in effluents discharged from the sewage treatment plant located along the bank of the Uji River, one tributary of the Yodo River system. Water samples were collected at the outlet of the sewage treatment plant for 16 consecutive days in May 1999 and 11 consecutive days in December 1999. Organic constituents were obtained via sorption to blue rayon and subsequent methanol elution. Extract mutagenic activity was measured using Salmonella typhimurium YG1024 with metabolic activation. PBTA-type mutagens (PBTA-1, PBTA-2, PBTA-3, PBTA-4, PBTA-5 and PBTA-6) were quantified by HPLC with electrochemical detection, followed by HPLC purification on reverse-phase columns. The study showed that PBTA-2, PBTA-3, PBTA-4 and PBTA-6 were detected in most samples. The total contribution of these four PBTA-type mutagens to overall extract mutagenicity is on average 33% for the May 1999 sample and 58% for the December 1999 sample. The individual PBTA compounds that had the largest contribution to the overall mutagenicity were PBTA-3 and PBTA-4, accounting for 11 and 16% in May 1999, and 25 and 26% in December 1999. A further comparative study was done in December 1999 using the blue rayon hanging method and the results were similar to those obtained using the blue rayon column method. In conclusion, the present study showed that PBTA-2, PBTA-3, PBTA-4 and PBTA-6 were commonly discharged from a sewage treatment plant into the Uji River, and they accounted for a substantial portion of the effluent mutagenicity.     

Absence of superoxide dismutase activity in a soluble cellular isoform of prion protein produced by baculovirus expression system

A method for expression and purification of a soluble form of histidine (HIS)-tagged murine prion protein (bacMuPrP), which lacks the entire C-terminal cleavage and glycosyl phosphatidyl inositol (GPI) addition site, has been developed using a recombinant baculovirus expression system and purification with Ni-NTA agarose affinity chromatography. In mammalian sources, PrP(C) is attached to the cell membrane by a GPI anchor. However, in our system, bacMuPrP was secreted into the media, enabling its easy purification in abundance. Indirect immunofluorescence studies and immunoblot analysis localized not in cell membrane but in the perinuclear endoplasmic reticulum region in cells and is secreted into the media. Tunicamycin treatment revealed non-glycosylated proteins were secreted into the media, suggesting that glycosylation is not necessary for bacMuPrP secretion. Density-gradient sedimentation analysis demonstrated a sedimentation coefficient of secretory bacMuPrP as 2.3 S, indicating a monomeric form. Although affinity-purified PrP from mouse brain or recombinant prion protein (PrP) produced by Escherichia coli and refolded in the presence of copper has been reported to display superoxide dismutase (SOD) activity, bacMuPrP did not show SOD activity. These results suggest that bacMuPrP has a different biochemical and biophysical characterization from mammalian and bacterial-derived PrP. Furthermore, this simple expression system may provide an adequate source for structural, functional, and biochemical analyses of PrP.     

Identification of a SNARE protein required for vacuolar protein transport in Schizosaccharomyces pombe

Intracellular vesicle trafficking is mediated by a set of SNARE proteins in eukaryotic cells. Several SNARE proteins are required for vacuolar protein transport and vacuolar biogenesis in Saccharomyces cerevisiae. A search of the Schizosaccharomyces pombe genome database revealed a total of 17 SNARE-related genes. Although no homologs of Vam3p, Nyv1p, and Vam7p have been found in S. pombe, we identified one SNARE-like protein that is homologous to S. cerevisiae Pep12p. However, the disruptants transport vacuolar hydrolase CPY (SpCPY) to the vacuole normally, suggesting that the Pep12 homolog is not required for vacuolar protein transport in S. pombe cells. To identify the SNARE protein(s) involved in Golgi-to-vacuole protein transport, we have deleted four SNARE homolog genes in S. pombe. SpCPY was significantly missorted to the cell surface on deletion of one of the SNARE proteins, Fsv1p (SPAC6F12.03c), with no apparent S. cerevisiae ortholog. In addition, sporulation, endocytosis, and in vivo vacuolar fusion appear to be normal in fsv1Delta cells. These results showed that Fsv1p is mainly involved in vesicle-mediated protein transport between the Golgi and vacuole in S. pombe cells.