Expression and glycosylation with polylactosamine of CD44 antigen on macrophages during follicular atresia in pig ovaries

Macrophages are essential in cleaning up apoptotic debris during follicular atresia. However, the key factors of this process are still unclear. In the present study, we evaluated CD44 mRNA, CD44 protein, and CD44 antigen glycosylation on macrophages during follicular atresia in the pig. Atresia was classified into five stages: stage I, healthy follicles; stage II, early atretic follicles having apoptotic granulosa cells with an unclear basement membrane; stage III, progressing atretic follicles having apoptotic granulosa cells completely diffused from the basement membrane; stage IV, late atretic follicles with increasing lysosomal activity; and stage V, disintegrated atretic follicles having collapsed theca cells and strong lysosomal activity. Immunohistological analysis showed that macrophages expressing CD44 invaded the inside of stage III follicles, accompanied by a collapse of basement membrane. Semiquantitative RT-PCR showed that only mRNA of the CD44 standard isoform (CD44s) was present in inner cells of follicles, and not any CD44 variant isoform (CD44v) mRNAs. The amount of CD44s mRNA was increased at stage III. Western blot and lectin blot analyses showed that CD44 was markedly expressed at stage III and glycosylated with polylactosamine at the same time. After macrophages invaded atretic follicles at stages III-V, the CD44 expressed on macrophages was glycosylated with polylactosamine. The lysosomal activity began to increase at stage IV, and reached the highest level at stage V. Increased CD44s protein and posttranslational modification of CD44 with polylactosamine on macrophages from stage III could be involved in the cleaning up apoptotic granulosa cells.     

The crystal structure of (S)-3-O-geranylgeranylglyceryl phosphate synthase reveals an ancient fold for an ancient enzyme

We report crystal structures of the citrate and sn-glycerol-1-phosphate (G1P) complexes of (S)-3-O-geranylgeranylglyceryl phosphate synthase from Archaeoglobus fulgidus (AfGGGPS) at 1.55 and 2.0 A resolution, respectively. AfGGGPS is an enzyme that performs the committed step in archaeal lipid biosynthesis, and it presents the first triose phosphate isomerase (TIM)-barrel structure with a prenyltransferase function. Our studies provide insight into the catalytic mechanism of AfGGGPS and demonstrate how it selects for the sn-G1P isomer. The replacement of "Helix 3" by a "strand" in AfGGGPS, a novel modification to the canonical TIM-barrel fold, suggests a model of enzyme adaptation that involves a "greasy slide" and a "swinging door." We propose functions for the homologous PcrB proteins, which are conserved in a subset of pathogenic bacteria, as either prenyltransferases or being involved in lipoteichoic acid biosynthesis. Sequence and structural comparisons lead us to postulate an early evolutionary history for AfGGGPS, which may highlight its role in the emergence of Archaea.     

Apoptotic process of porcine intestinal M cells

Membranous (M) cells of the follicle-associated epithelium (FAE) are believed to sample antigens from the gut lumen. However, the origin, differentiation mechanism, and cell death of M cells are still a matter of controversy. Therefore, we investigated the process of M cell differentiation and determined their fate in the intestine of three-way crossbred female pigs. We used anti-cytokeratin 18 and anti-PCNA antibodies to distinguish M cells and proliferative cells and performed immunohistochemistry, enzyme histochemistry, and scanning electron microscopy on fresh ileal Peyer’s patches. Cell migration and apoptotic cells were detected by BrdU labeling and the TUNEL method, respectively. The turnover of the FAE was similar to that of the villi. M cells were mostly observed from the FAE crypt to the FAE periphery, but not in the FAE apex. As proliferative M cells (cytokeratin 18⁺/PCNA⁺ cells) have previously been detected in the FAE crypt, porcine M cells may be directly derived from intestinal epithelial stem cells and committed as a distinct cell lineage in the crypts. M cells from the FAE periphery were unstained or only weakly stained for alkaline phosphatase, whereas cytokeratin 18⁺/alkaline phosphatase⁺ cells lying near to the FAE apex showed a columnar shape similar to that of adjacent enterocytes. These data suggest that the committed M cells differentiate to mature M cells by contact with lymphocytes at the FAE periphery, and that they trans-differentiate to enterocytes and are finally excluded near the FAE apex. This investigation was supported by a Grant-in-Aid for Scientific Research (16658107) from the Ministry of Education, Culture, Sports, Science and Technology, by two grants (Prion Project and Secure and Healthy Livestock Farming Project) from the Ministry of Agriculture, Forestry and Fisheries, and by a grant from the Naito Foundation.     

Substrate specificity of thermostable D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589

D-Alanine-D-alanine ligase (Ddl) and its mutants maintain the biosynthesis of peptidoglycan, and the substrate specificity of Ddls partially affects the resistance mechanism of vancomycin-resistant enterococci. Through investigation of Ddls, Ddl from Thermotoga maritima ATCC 43589 showed novel characteristics, vis. thermostability up to 90 degrees C and broad substrate specificity toward 15 D-amino acids, particularly D-alanine, D-cysteine, and D-serine, in that order.     

Rapid evaluation of the efficacy of microbial cell removal from fabrics

The efficacy of microbial cell removal (EMR) from fabrics is a practically important indicator for the evaluation of cleansers and detergents. EMR is expressed quantitatively by the relative number of viable cells remaining on a fabric swatch after the treatment with these reagents. In order to count the viable cells on the swatch directly and rapidly, we have developed a unique microscopic imaging system with an ultra-deep focusing range. Standard swatches of cotton fabric were inoculated with microorganisms such as Pseudomonas fluorescence, Staphylococcus aureus, or Candida albicans. After the incubation on an agar medium, each swatch was treated with a fluorescent glucose, 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxyglucose, to stain only viable cells. The images of every cell distributed within the surface layer with no greater than 130 μm thickness could be integrated into one image. Thus visualized cells could be counted automatically by a novel imaging program. Using a pair of cotton swatches (0.5×1.0 cm²) inoculated with C. albicans, EMR was evaluated quantitatively. Before washing, the total number of viable cells found on the observation area (3.8×10⁻⁴ cm²) was 288 cells. After washing with a test detergent, no cell (<1) was detected. For this case, EMR was given by the formula: log(288/<1)=greater than 2.5. The imaging and cell count of a test fabric could be performed within 1 h.     

N-acetylcysteine attenuates TNF-alpha induced changes in secretion of interleukin-6, plasminogen activator inhibitor-1 and adiponectin from 3T3-L1 adipocytes

TNF-alpha is a key molecule in obesity-related metabolic disturbances. This study was designed to determine whether N-acetylcysteine (NAC), an antioxidant, prevents the activation of nuclear factor-kappaB (NF-kappaB) by exogenously administered TNF-alpha in adipocytes, and whether such change affects the production of adipocytokines. The treatment of well-differentiated 3T3-L1 cells with 20 mM of NAC significantly increased the reduced glutathione concentration up to 150% of control. The treatment with 10 ng/ml of TNF-alpha decreased antioxidant enzyme levels such as CuZn-superoxide dismutase (SOD), MnSOD and catalase, and activated NF-kappaB in 3T3-L1 adipocytes. The activation of NF-kappaB was significantly prevented by the pretreatment with 20 mM of NAC. TNF-alpha (1-10 ng/ml) dose-dependently increased interleukin (IL)-6 and plasminogen activator inhibitor-1 (PAI-1) secretion from 3T3-L1 adipocytes, while decreased adiponectin secretion. NAC (5-20 mM) attenuated the TNF-alpha-induced changes in these adipocytokine secretions in a dose-dependent manner. The effect of TNF-alpha and NAC on the adipocytokine productions was exerted at the m-RNA level, judging from results of the real time RT-PCR analysis. The present study revealed that NAC inhibited the TNF-alpha-mediated activation of NF-kappaB and improved the adverse changes in the levels of IL-6, PAI-1 and adiponectin in 3T3-L1 adipocytes. NAC may have the potential to improve the obesity-related abnormal adipocytokine metabolism by attenuating the TNF-alpha-induced oxidant-antioxidant imbalance in adipocytes.     

Transcytosis of Murine-Adapted Bovine Spongiform Encephalopathy Agents in an In Vitro Bovine M Cell Model

Transmissible spongiform encephalopathies (TSE), including bovine spongiform encephalopathy (BSE), are fatal neurodegenerative disorders in humans and animals. BSE appears to have spread to cattle through the consumption of feed contaminated with BSE/scrapie agents. In the case of an oral infection, the agents have to cross the gut-epithelial barrier. We recently established a bovine intestinal epithelial cell line (BIE cells) that can differentiate into the M cell type in vitro after lymphocytic stimulation (K. Miyazawa, T. Hondo, T. Kanaya, S. Tanaka, I. Takakura, W. Itani, M. T. Rose, H. Kitazawa, T. Yamaguchi, and H. Aso, Histochem. Cell Biol. 133:125-134, 2010). In this study, we evaluated the role of M cells in the intestinal invasion of the murine-adapted BSE (mBSE) agent using our in vitro bovine intestinal epithelial model. We demonstrate here that M cell-differentiated BIE cells are able to transport the mBSE agent without inactivation at least 30-fold more efficiently than undifferentiated BIE cells in our in vitro model. As M cells in the follicle-associated epithelium are known to have a high ability to transport a variety of macromolecules, viruses, and bacteria from gut lumen to mucosal immune cells, our results indicate the possibility that bovine M cells are able to deliver agents of TSE, not just the mBSE agent.Transmissible spongiform encephalopathies (TSE) or prion diseases, including human Creutzfeldt-Jakob disease (CJD) and endemic sheep scrapie, are fatal neurodegenerative diseases. The host cellular prion protein (PrP^C), which is thought to have neuroprotective function, is expressed in both humans and a range of other animal species (36), and PrP^C expression is essential for TSE disease susceptibility (7). The prion hypothesis suggests that infectious abnormally folded prion protein (PrP^Sc) is the primary or sole composition of the infectious agent of TSE (known as the prion). However, the molecular composition of PrP^Sc remains speculative and unclear. It is well known that the detergent-insoluble and relatively proteinase K (PK)-resistant prion protein (PrP-res) is detectable in many kinds of TSE-infected tissues, including the brain. Although some studies have revealed that PrP-res does not correlate with infectivity levels in animal tissues as well as in subcellular fractions (37, 40), PrP-res is a useful surrogate marker for TSE infection.Bovine spongiform encephalopathy (BSE) is a TSE of cattle. The first case of BSE in the world was found in the United Kingdom in 1986 (41), and it spread to continental Europe, North America, and Japan. At present, BSE is a threat to human health because of the appearance of BSE-linked variant Creutzfeldt-Jakob disease (vCJD). The cattle BSE agent appears to spread to the cattle population through the consumption of rendered meat and bone meal contaminated with BSE-infected brain or spinal cord (32). Likewise, the transmission of vCJD to humans is likely to have occurred following the consumption of BSE-contaminated food (6, 13, 45). In cases of oral transmissions such as BSE and vCJD, TSE agents first have to cross the gut epithelium, but the exact mechanisms for intestinal invasion still are unknown.Intestinal epithelial cells are bound to each other by tight junctions. This close-packed structure forms a highly selective barrier for macromolecules and limits the access of pathogenic bacteria to the underlying host tissues (43). Gut epithelia are composed of two different epithelial types. One is the villous epithelium, and the other is the follicle-associated epithelium (FAE), which overlies gut-associated lymphoid tissues (GALTs) such as Peyer''s patches. The FAE is considerably different from the surrounding villous epithelium, in that it contains membranous (M) cells. Because M cells have a high capacity for the transcytosis of a wide range of macromolecules, viruses, and microorganisms, they are specialized epithelial cells and act as an antigen sampling system from the gut lumen (28). M cells are, however, exploited by some pathogenic microorganisms and viruses as the entry site to invade the body (20, 29). In fact, some experiments have proposed that M cells transport TSE agents (12) and that Peyer''s patches including the FAE are associated with TSE disease susceptibility (35). In contrast, some authors have suggested the M cell-independent pathway as the main transport route of TSE agents across the intestinal epithelium (16, 23, 27). The intestinal cell types involved in the transport of TSE agents therefore are still a matter of controversy at this stage.Recently, we succeeded in the establishment of a bovine intestinal epithelial cell line (BIE cells) and the development of an in vitro bovine M cell model by coculture with murine intestinal lymphocytes or the supernatant of bovine peripheral blood mononuclear cells (PBMC) stimulated by interleukin 2 (IL-2) (25). In this study, we investigate whether M cells can transport the murine-adapted BSE (mBSE) agent using BIE cells. We demonstrate here that M cell-differentiated BIE cells are able to deliver mBSE agents at least 30-fold more efficiently than undifferentiated BIE cells, although a small number of the mBSE agents pass through undifferentiated BIE cells. Our findings thus provide an insight into the uptake mechanisms of TSE agents, including the cattle BSE agent from the gut lumen.     

Potential Roles of CCR5+ CCR6+ Dendritic Cells Induced by Nasal Ovalbumin plus Flt3 Ligand Expressing Adenovirus for Mucosal IgA Responses

We assessed the role of CCR5⁺/CCR6⁺/CD11b⁺/CD11c⁺ dendritic cells (DCs) for induction of ovalbumin (OVA)-specific antibody (Ab) responses following mucosal immunization. Mice given nasal OVA plus an adenovirus expressing Flt3 ligand (Ad-FL) showed early expansion of CCR5⁺/CCR6⁺/CD11b⁺/CD11c⁺ DCs in nasopharyngeal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLNs). Subsequently, this DC subset became resident in submandibular glands (SMGs) and nasal passages (NPs) in response to high levels of CCR-ligands produced in these tissues. CD11b⁺/CD11c⁺ DCs were markedly decreased in both CCR5^−/− and CCR6^−/− mice. Chimera mice reconstituted with bone marrow cells from CD11c-diphtheria toxin receptor (CD11c-DTR) and CCR5^−/− or CD11c-DTR and CCR6^−/− mice given nasal OVA plus Ad-FL had elevated plasma IgG, but reduced IgA as well as low anti-OVA secretory IgA (SIgA )Ab responses in saliva and nasal washes. These results suggest that CCR5⁺CCR6⁺ DCs play an important role in the induction of Ag-specific SIgA Ab responses.     

Kinematic study of root elongation in Arabidopsis thaliana with a novel image-analysis program

The measurement of the spatial profile of root elongation needs to determine matching points between time-lapse images and calculate their displacement. These data have been obtained by laborious manual methods in the past. Some computer-based programs have been developed to improve the measurement, but they require many time-series digital images or sprinkling graphite particles on the root prior to image capture. Here, we have developed GrowthTracer, a new image-analysis program for the kinematic study of root elongation. GrowthTracer employs a multiresolution image matching method with a nonlinear filter called the critical point filter (CPF), which extracts critical points from images at each resolution and can determine precise matching points by analysis of only two intact images, without pre-marking by graphite particles. This program calculates the displacement of each matching point and determines the displacement velocity profile along the medial axis of the root. In addition, a manual input of distinct matching points increases the matching accuracy. We show a successful application of this novel program for the kinematic analysis of root growth in Arabidopsis thaliana.     

Effect of obesity and troglitazone on expression of two glutathione peroxidases: Cellular and extracellular types in serum,kidney and adipose tissue

To determine the effect of obesity on expression of cellular- (C-) and extracellular (EC-) glutathione peroxidase (GPX) in serum, kidney and adipose tissue, we measured GPX in serum, kidneys and adipose tissue of the obese Otsuka-Long-Evans-Tokushima Fatty (OLETF) rat and its lean counterpart (LETO). We also investigated the effect of troglitazone. Five each of OLETF and LETO rats were fed diet with or without 0.2% troglitazone for 10 days. Final body weight, kidney weight, blood glucose and serum tumor necrosis factor-α (TNF-α) level were higher in OLETF rats than in LETO rats. Serum and kidney GPX activities were higher, but adipose tissue GPX activity was lower, in OLETF rats than in LETO rats. Troglitazone treatment decreased adipose tissue GPX activity and abolished overproduction of TNF-α in OLETF rats. Immunoblot analysis, for the first time, revealed that both obesity and troglitazone suppressed the protein signals for C-GPX and EC-GPX in adipose tissue. Serum protein carbonyl groups were increased in OLETF rats and troglitazone completely blocked this increase. Increased serum GPX activity in obese rat was due to the increased secretion of EC-GPX from the kidney. Troglitazone protected against the enhanced oxidative stress induced by obesity independently of the serum GPX concentration.