Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells

In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein-tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.     

Ontogenetic wood anatomy of tree and subtree species of Nepalese Rhododendron (Ericaceae) and characterization of shrub species

Ontogenetic trends in the wood structure of Nepalese Rhododendron were studied in 15 specimens of two tree and four subtree species. Average growth ring width was constant from pith to bark in spite of occurrences of extremely narrow, false, or discontinuous rings. Vessel density, vessel area, vessel element length, and multiseriate ray height generally had an initial increase or decrease to 1.5 cm radius and near plateau or slight decrease or increase outward. Multiseriate ray density and area percentage were variable between specimens without a clear pattern. Ontogenetic trends from pith to fully mature wood in trees plus subtrees were inferred by treating the measurements in the present study with those of mature individuals in a previous study. Comparison of trends in trees plus subtrees and those in shrubs lead to ecological or systematic groupings. Vessel features showed that alpine shrub species have distinctly small, numerous vessels composed of short vessel elements. Multiseriate ray features indicated a systematic difference between the trees plus subtrees of subgenus Hymenanthes and the shrubs of subgenus Rhododendron. Vessel features of alpine shrubs may be an adaptation against frequent freeze-thaw cycles or the result of growth stress imposed by the severe alpine environment.     

Association between a single-nucleotide polymorphism in the promoter of the human interleukin-3 gene and rheumatoid arthritis in Japanese patients, and maximum-likelihood estimation of combinatorial effect that two genetic loci have on susceptibility to the disease

Genetic variants of interleukin-3 (IL-3), a well-studied cytokine, may have a role in the pathophysiology of rheumatoid arthritis (RA); but reports on this association sometimes conflict. A case-control study was designed to investigate association between RA and a single-nucleotide polymorphism (SNP) in the IL-3 promoter region. Comparison of cases of RA versus control individuals yielded a chi(2) value of 14.28 (P=.0002), with a genotype odds ratio of 2.24 (95% confidence interval [95%CI] 1.44-3.49). When female cases with earlier onset were compared with female control individuals, the SNP revealed an even more significant correlation, with chi2=21.75 (P=.000004) and a genotype odds ratio of 7.27 (95%CI 2.80-18.89). The stronger association that we observed in this clinically distinct subgroup (females with early onset), within a region where linkage disequilibrium was not significantly extended, suggested that the genuine RA locus should locate either within or close to the IL-3 gene. Combined genotype data on SNPs on eight other candidate genes were combined with our IL-3 results, to estimate relationships between pairs of loci and RA, by maximum-likelihood analysis. The utility of combining the genotype data in this way to identify possible contributions of various genes to this disease is discussed.     

Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump

Biliary excretion of certain bile acids is mediated by multidrug resistance associated protein 2 (Mrp2) and the bile salt export pump (Bsep). In the present study, the transport properties of several bile acids were characterized in canalicular membrane vesicles (CMVs) isolated from Sprague--Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR) whose Mrp2 function is hereditarily defective and in membrane vesicles isolated from Sf9 cells infected with recombinant baculovirus containing cDNAs encoding Mrp2 and Bsep. ATP-dependent uptake of [(3)H]taurochenodeoxycholate sulfate (TCDC-S) (K(m)=8.8 microM) and [(3)H]taurolithocholate sulfate (TLC-S) (K(m)=1.5 microM) was observed in CMVs from SD rats, but not from EHBR. In addition, ATP-dependent uptake of [(3)H]TLC-S (K(m)=3.9 microM) and [(3)H]taurocholate (TC) (K(m)=7.5 microM) was also observed in Mrp2- and Bsep-expressing Sf9 membrane vesicles, respectively. TCDC-S and TLC-S inhibited the ATP-dependent TC uptake into CMVs from SD rats with IC(50) values of 4.6 microM and 1.2 microM, respectively. In contrast, the corresponding values for Sf9 cells expressing Bsep were 59 and 62 microM, respectively, which were similar to those determined in CMVs from EHBR (68 and 33 microM, respectively). By co-expressing Mrp2 with Bsep in Sf9 cells, IC(50) values for membrane vesicles from these cells shifted to values comparable with those in CMVs from SD rats (4.6 and 1.2 microM). Moreover, in membrane vesicles where both Mrp2 and Bsep are co-expressed, preincubation with the sulfated bile acids potentiated their inhibitory effect on Bsep-mediated TC transport. These results can be accounted for by assuming that the sulfated bile acids trans-inhibit the Bsep-mediated transport of TC.     

Structure and function analysis of urinary trypsin inhibitor (UTI): identification of binding domains and signaling property of UTI by analysis of truncated proteins

The binding of urinary trypsin inhibitor (UTI) to its binding sites/receptors on tumor cells inhibits cell invasion in a number of experimental systems and that UTI downregulates constitutive and phorbol ester-induced urokinase production by certain tumor cells. To determine whether the carbohydrate moieties and core protein are required for urokinase suppression, we obtained UTI derivatives that contained O-glycoside-linked N-terminal glycopeptide (UTIm1), N-glycoside-linked C-terminal tandem Kunitz domains (UTIm2), UTI lacking O-glycoside (UTIc), asialo UTI (UTIa), UTI lacking N-glycoside (UTIn), purified Kunitz domain II of UTI (HI-8), and recombinant Kunitz domain II of UTI (R-020). The IC(50) of inhibiting binding of (125)I-labeled UTI to cells was indistinguishable for UTIa, UTIn and intact UTI, whereas the IC(50) for inhibiting binding of (125)I-labeled UTI to cells was 2.5-, 25- and 29-fold greater for UTIm1, UTIm2 and UTIc than for native UTI. We next looked at the suppression of the urokinase expression by UTI derivatives. An enzyme-linked immunosorbent assay was carried out to measure secreted and cell-associated urokinase. Intact UTI, UTIa, or UTIn effectively suppressed urokinase expression, but UTIm1, UTIm2, UTIc, HI-8 and R-020 had no significant effect. These data show that UTI requires either the N-terminal extension with the O-linked carbohydrate moiety (chondroitin 4-sulfate sugar side chain; Ala1 to Lys21 residues) or the Kunitz domain I (Lys22 to Arg77 residues) of UTI to bind to cells, but the urokinase expression was inhibited only by the O-glycoside-linked core protein without the N-glycoside side chain.     

Gonadotropin-releasing hormone in third ventricular cerebrospinal fluid of the heifer during the estrous cycle

The release profile of GnRH in cerebrospinal fluid (CSF) and its correlation with LH in peripheral blood of ovary-intact heifers during the estrous cycle were investigated. A silicon catheter was placed into the third ventricle of six heifers using ultrasonography. During the mid-luteal phase, the heifers were injected with prostaglandin F(2alpha) to induce luteolysis. Surges of CSF GnRH (66.7 h after prostaglandin F(2alpha) administration) and peripheral LH (66.3 h) occurred simultaneously and were coincident with the onset of estrus (67.0 h). Duration of elevated GnRH concentration considerably overlapped with the estrous phase in each of the heifers. Mean pulse frequencies of both GnRH and LH were significantly higher during the proestrous and early luteal phases than during the mid-luteal phase, while mean concentration and pulse amplitude of both GnRH and LH were not different between these three phases. Of all the GnRH pulses identified, more than 80% were accompanied by an LH pulse during the proestrous and early luteal phases. However, the proportion of GnRH pulses that were coincident with an LH pulse during the mid-luteal phase decreased to 60%. The results clearly demonstrate that a dynamic (pulse) and longer-term (surge) changes of GnRH release into CSF are physiologically expressed during the estrous cycle in heifers, and the pattern of pulsatile GnRH secretion in heifers depends upon their estrous cycle.     

Degradation of bisphenol A by purified laccase from Trametes villosa

Degradation of bisphenol A (BPA), an endocrine-disrupting chemical, was studied with a purified laccase from the basidiomycete Trametes villosa. SDS--polyacrylamide gel electrophoresis of the purified laccase gave one single band with a mobility corresponding to MW 65 kDa. The absorption spectrum showed the characteristics of a blue copper protein with a maximum peak at 600 nm. HPLC analysis revealed that 2.2 micromol BPA were degraded by incubation with 1.5 units of the purified laccase in a total volume of 1.0 ml at pH 6.0 and 60 degrees C for 3 h. The enzyme reaction proceeded rapidly without requirement of mediators for the electron transfer. Isolation and identification of several reaction products are in progress, in which one product was identified as 4-isopropenylphenol by a gas chromatography--mass spectrophotometer.     

The PUB domain: a putative protein-protein interaction domain implicated in the ubiquitin-proteasome pathway

Cytoplasmic peptide:N-glycanase (PNGase) is a de-N-glycosylating enzyme which may be involved in the proteasome-dependent pathway for degradation of misfolded glycoproteins formed in the endoplasmic reticulum (ER) that are exported into the cytoplasm. A cytoplasmic PNGase found in Saccharomyces cerevisiae, Png1p, is widely distributed in higher eukaryotes as well as in yeast (Suzuki, T., et al. J. Cell Biol. 149, 1039-1051, 2000). The recently uncovered complete genome sequence of Arabidopsis thaliana prompted us to search for the protein homologue of Png1p in this organism. Interestingly, when the mouse Png1p homologue sequence was used as a query, not only a Png1p homologue containing a transglutaminase-like domain that is believed to contain a catalytic triad for PNGase activity, but also four proteins which had a domain of 46 amino acids in length that exhibited significant similarity to the N-terminus of mouse Png1p were identified. Moreover, three of these homologous proteins were also found to possess a UBA or UBX domain, which are found in various proteins involved in the ubiquitin-related pathway. We name this newly found homologous region the PUB (Peptide:N-glycanase/UBA or UBX-containing proteins) domain and propose that this domain may mediate protein-protein interactions.     

Exposure of RBL-2H3 mast cells to Ag(+) induces cell degranulation and mediator release

There is a growing need to understand the impact of environmental sulfhydryl group-reactive heavy metals on the immune system. Here we show that Ag(+) induces mast cell degranulation, as does the aggregation of the high affinity immunoglobulin E receptor (FcepsilonRI). Micromolar quantities of Ag(+) specifically induced degranulation of mast cell model rat basophilic leukemia (RBL-2H3) cells without showing cytotoxicity. The Ag(+)-mediated degranulation could be observed as rapidly as 5 min after the addition of the ions. Ag(+) also induced a rapid change in tyrosine phosphorylation of multiple cellular proteins including the focal adhesion kinase but not Syk kinase. The Syk-selective inhibitor piceatannol and the Src family-selective tyrosine kinase inhibitor PP1 dose-dependently inhibited FcepsilonRI-mediated degranulation, whereas neither compound inhibited the Ag(+)-mediated degranulation. Furthermore, likewise FcepsilonRI aggregation, Ag(+) also induced leukotriene secretion. These results show that Ag(+) activates RBL-2H3 mast cells through a tyrosine phosphorylation-linked mechanism, which is distinct from that involved in FcepsilonRI-mediated activation.