MHC class I-like MILL molecules are beta2-microglobulin-associated, GPI-anchored glycoproteins that do not require TAP for cell surface expression

MILL (MHC class I-like located near the leukocyte receptor complex) is a family of MHC class I-like molecules encoded outside the MHC, which displays the highest sequence similarity to human MICA/B molecules among known class I molecules. In the present study, we show that the two members of the mouse MILL family, MILL1 and MILL2, are GPI-anchored glycoproteins associated with beta2-microglobulin (beta2m) and that cell surface expression of MILL1 or MILL2 does not require functional TAP molecules. MILL1 and MILL2 molecules expressed in bacteria could be refolded in the presence of beta2m, without adding any peptides. Hence, neither MILL1 nor MILL2 is likely to be involved in the presentation of peptides. Immunohistochemical analysis revealed that MILL1 is expressed in a subpopulation of thymic medullary epithelial cells and a restricted region of inner root sheaths in hair follicles. The present study provides additional evidence that MILL is a class I family distinct from MICA/B.     

PTEN is involved in the signal transduction pathway of contact inhibition in endometrial cells

PTEN is involved in the regulation of normal cellular functions in addition to its well–known role as a tumor suppressor. In the present study, we have shown that stable transfection of the PTEN gene into PTEN–mutated endometrial carcinoma cells leads to contact inhibition accompanied by a decreased level of phosphorylated–Akt (p–Akt) expression, an increase in p27^Kip1, and a decrease in β–catenin. PTEN–induced cells with contact inhibition exhibit G0–G1 cell-cycle arrest, and the Ki–67 labeling index is reduced. These changes are canceled by transfection of a double–stranded short–interfering RNA against the PTEN gene. Normal endometrial stromal cells increase their PTEN expression when reaching confluence; this is followed by changes in the expression of Akt–related proteins in the same way as in tumor cells. These results indicate that PTEN, p–Akt, p27, and β–catenin are involved in the signal transduction of contact inhibition and suggest that PTEN may, in part, control the proliferation of endometrial carcinoma cells through the induction of contact inhibition.     

Control of isoperoxidases involved in chlorophyll degradation of stored broccoli (Brassica oleracea) florets by heat treatment

Changes in isoperoxidases involved in chlorophyll (Chl) degradation of stored broccoli (Brassica oleracea L.) florets and their control by heat treatment (HT) were determined. Chl a and b contents in non-heat-treated broccoli florets decreased greatly after 2 days at 15 degrees C, whereas the contents in heat-treated florets (50 degrees C for 2 h) showed almost no change. Three isoperoxidases involved in Chl degradation were detected by means of molecular exclusion chromatography and the molecular weights of those isoperoxidases were about 95 (Type I), 67 (Type II) and 56 (Type III) kDa, respectively. Only Type I was detected in broccoli florets immediately after harvest, and its activity in non-heat-treated broccoli increased greatly during storage. Both Type II and Type III were present in non-heat-treated broccoli with floret senescence. HT suppressed the enhancement of all of the isoperoxidase activities. Cycloheximide treatment also effectively retarded the increase in Types I, II and III isoperoxidase activities concomitant with the suppression of floret yellowing. The K(m) values corresponding to Chl a of Type II and Type III were lower than Type I, and the V(max)/K(m) values corresponding to Chl a of Type II and Type III were higher than Type I. This suggests that both Types II and III could be closely associated with Chl degradation in broccoli florets and that HT might inhibit floret senescence by suppression of isoperoxidase activities.     

Cytoskeleton alterations induced by Geodia corticostylifera depsipeptides in breast cancer cells

Crude extracts of the marine sponge Geodia corticostylifera from Brazilian Coast have previously shown antibacterial, antifungal, cytotoxic, haemolytic and neurotoxic activities. The present work describes the isolation of the cyclic peptides geodiamolides A, B, H and I (1-4) from G. corticostylifera and their anti-proliferative effects against sea urchin eggs and human breast cancer cell lineages. Its structure-activity relationship is discussed as well. In an initial series of experiments these peptides inhibited the first cleavage of sea urchin eggs (Lytechinus variegatus). Duplication of nuclei without complete egg cell division indicated the mechanism of action might be related to microfilament disruption. Further studies showed that the geodiamolides have anti-proliferative activity against human breast cancer cell lines (T47D and MCF7). Using fluorescence techniques and confocal microscopy, we found evidence that the geodiamolides A, B, H and I act by disorganizing actin filaments of T47D and MCF7 cancer cells, in a way similar to other depsipeptides (such as jaspamide 5 and dolastatins), keeping the normal microtubule organization. Normal cells lines (primary culture human fibroblasts and BRL3A rat liver epithelial cells) were not affected by the treatment as tumor cells were, thus indicating the biomedical potential of these compounds.     

Synthesis and enzymatic susceptibility of a series of novel GM2 analogs

A series of GM2 analogs in which GM2 epitope was coupled to a variety of glycosyl lipids were designed and synthesized to investigate the mechanism of enzymatic hydrolysis of GM2 ganglioside. The coupling of N-Troc-protected sialic acid and p-methoxyphenyl galactoside acceptor gave the crystalline disaccharide, which was further coupled with galactosamine donor to give the desired GM2 epitope trisaccharide. After conversion into the corresponding glycosyl donor, the trisaccharide was coupled with galactose, glucose and artificial ceramide (B30) to give the final compounds. The result on hydrolysis of GM2 analogs indicates that GM2 activator protein requires one spacer sugar between GM2 epitope and the lipid moiety to assist the hydrolysis of the terminal GalNAc residue. Synthetic studies on sialoglycoconjugates, Part 140. For part 139, see Ref [1].     

Acidianus manzaensis sp. nov., a Novel Thermoacidophilic Archaeon Growing Autotrophically by the Oxidation of H2 with the Reduction of Fe3+

A novel thermoacidophilic iron-reducing Archaeon, strain NA−1, was isolated from a hot fumarole in Manza, Japan. Strain NA-1 could grow autotrophically using H₂ or S⁰ as an electron donor and Fe³⁺ as an electron acceptor, and also could grow heterotrophically using some organic compounds. Fe³⁺ and O₂ served as electron acceptors for growth. However, S⁰, NO₃ ⁻, NO₂ ⁻, SO₄ ²⁻, Mn⁴⁺, fumarate, and Fe₂O₃ did not serve as electron acceptors. The ranges of growth temperature and pH were 60–90°C (optimum: 80°C) and pH 1.0–5.0 (optimum: pH 1.2–1.5), respectively. Cells were nearly regular cocci with an envelope comprised of the cytoplasmic membrane and a single outer S-layer. The crenarchaeal-specific quinone (cardariellaquinone) was detected, and the genomic DNA G + C content was 29.9 mol%. From 16S rDNA analysis, it was determined that strain NA-1 is closely related to Acidianus ambivalens (93.1%) and Acidianus infernus (93.0%). However, differences revealed by phylogenetic and phenotypic analyses clearly show that strain NA-1 represents a new species, Acidianus manzaensis, sp. nov., making it the first identified thermoacidophilic iron-reducing microorganism (strain NA-1^T = NBRC 100595 = ATCC BAA 1057). Strain NA-1 has been deposited in the culture collections of the National Institute of Technology and Evolution (NBRC 100595) and American Type Culture Collection (ATCC BAA 1057). The 16S rDNA sequence has been deposited at GenBank under accession number AB182498.     

Evolution and homology of the astragalus in early amniotes: new fossils, new perspectives

The reorganization of the ankle in basal amniotes has long been considered a key innovation allowing the evolution of more terrestrial and cursorial behavior. Understanding how this key innovation arose is a complex problem that largely concerns the homologizing of the amniote astragalus with the various ossifications in the anamniote tarsus. Over the last century, several hypotheses have been advanced homologizing the amniote astragalus with the many ossifications in the ankle of amphibian-grade tetrapods. There is an emerging consensus that the amniote astragalus is a complex structure emerging via the co-ossification of several originally separate elements, but the identities of these elements remain unclear. Here we present new fossil evidence bearing on this contentious question. A poorly ossified, juvenile astragalus of the large captorhinid Moradisaurus grandis shows clear evidence of four ossification centers, rather than of three centers or one center as posited in previous models of astragalus homology. Comparative material of the captorhinid Captorhinikos chozaensis is also interpretable as demonstrating four ossification centers. A new, four-center model for the homology of the amniote astragalus is advanced, and is discussed in the context of the phylogeny of the Captorhinidae in an attempt to identify the developmental transitions responsible for the observed pattern of ossification within this clade. Lastly, the broader implications for amniote phylogeny are discussed, concluding that the neomorphic pattern of astragalus ossification seen in all extant reptiles (including turtles) arose within the clade Diapsida.     

Specific binding of CO to tetraheme cytochrome c3

Carbon monoxide (CO) has been identified as another bioactive molecule like NO. Binding of CO to a tetraheme cytochrome c(3) (cyt c(3)) was investigated using visible absorption spectroscopy, circular dichroism (CD), and NMR. CO was found to bind to the four hemes in different manners. CD spectra, however, indicated that only single-site CO binding can keep the protein intact. The K(d) for the single-site binding was 8.0 microM, which is a typical value for a CO sensor protein. Furthermore, NMR spectra of uniformly (15)N-labeled and specifically [(15)N]His-labeled proteins have provided evidence that CO specifically binds to the sixth coordination site of heme 2 via single-site binding. The CO-bound cyt c(3) could conduct redox reactions. In light of triheme cytochrome c(7), the CO-bound cyt c(3) may work as an electron transporter. It was reported for sulfate-reducing bacteria that CO can be used as an energy source and CO cycling is operating like H(2) cycling. Therefore, the CO-bound cyt c(3) may play a role in maintaining electron transport pathways on accumulation of toxic CO for its utilization.     

Peroxisome proliferator-activated receptor alpha-independent peroxisome proliferation

Hepatic peroxisome proliferation, increases in the numerical and volume density of peroxisomes, is believed to be closely related to peroxisome proliferator-activated receptor alpha (PPARalpha) activation; however, it remains unknown whether peroxisome proliferation depends absolutely on this activation. To verify occurrence of PPARalpha-independent peroxisome proliferation, fenofibrate treatment was used, which was expected to significantly enhance PPARalpha dependence in the assay system. Surprisingly, a novel type of PPARalpha-independent peroxisome proliferation and enlargement was uncovered in PPARalpha-null mice. The increased expression of dynamin-like protein 1, but not peroxisome biogenesis factor 11alpha, might be associated with the PPARalpha-independent peroxisome proliferation at least in part.