Production of the Carotenoids Lycopene, β-Carotene, and Astaxanthin in the Food Yeast Candida utilis

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, β-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promoters and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, β-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.     

Immunocytochemistry of Rhamnogalacturonan II in Cell Walls of Higher Plants

A polyclonal antibody against a borate-RG-II complex is raisedin rabbits. The antibody recognized RG-II exclusively in cellwall polysaccharides. Immunocytochemical studies demonstratedthat the epitope is ubiquitous in cell walls of all the cellsin radish and rice roots, cultured tobacco cells, red cloverroot nodules, and lily growing pollen tubes. The label was denserin proximal to plasma membrane, and not detected in middle lamella,suggesting that borate may cross-link newly secreted pecticpolysaccharides at the membrane-cell wall interface. (Received October 13, 1997; Accepted February 16, 1998)     

Interferon Yield and MHC Antigen Expression of Human Medulloblastoma Cells and Its Suppression During Dibutyryl Cyclic AMP-Induced Differentiation: Do Medulloblastoma Cells Derive from Bipotent Neuronal and Glial Progenitors?

1. Human medulloblastoma (ONS-76), a central nervous system (CNS)-derived undifferentiated cell line, was found to possess glial characteristics as defined by responses in the interferon (IFN) system; ONS-76 cells produced as much IFN- as human fibroblast and glioma cells by viral infection and poly(I):poly(C) induction.2. Major histocompatibility complex (MHC) class I antigens were also induced under IFN- stimulation. ONS-76 cells expressed neurofilament protein, as shown by Northern blot analysis, and morphological differentiation was induced by dibutyryl cyclic AMP (dcAMP).3. Expression of IFN- and MHC class I antigens was suppressed in ONS-76 cells during the dcAMP-induced differentiation.4. These results showed that ONS-76 cells possessed a glial properly in IFN system responses and a neuronal property in cytoskeleton protein, suggesting that the precursors of medulloblastoma may be characterized as bipotent neuronal and glial progenitors in CNS.     

Polymorphisms Influencing Expression of Dermonecrotic Toxin in Bordetella bronchiseptica

Bordetella bronchiseptica is a pathogenic bacterium causing respiratory infections in a broad range of mammals. Recently, we determined the whole genome sequence of B. bronchiseptica S798 strain isolated from a pig infected with atrophic rhinitis and found four single-nucleotide polymorphisms (SNPs) at positions -129, -72, +22, and +38 in the region upstream of dnt encoding dermonecrotic toxin (DNT), when compared with a rabbit isolate, RB50. DNT is known to be involved in turbinate atrophy observed in atrophic rhinitis. Immunoblotting, quantitative real-time PCR, and β-galactosidase reporter assay revealed that these SNPs resulted in the increased promoter activity of dnt and conferred the increased ability to produce DNT on the bacteria. Similar or identical SNPs were also found in other pig isolates kept in our laboratory, all of which produce a larger amount of DNT than RB50. Our analysis revealed that substitution of at least two of the four bases, at positions -72 and +22, influenced the promoter activity for dnt. These results imply that these SNPs are involved in the pathogenicity of bordetellae specific to pig diseases.     

Abnormal Localization of STK17A in Bile Canaliculi in Liver Allografts: An Early Sign of Chronic Rejection

The biological significance of STK17A, a serine/threonine kinase, in the liver is not known. We analyzed STK17A expression in HepG2 cells and human liver tissue. Accordingly, we investigated whether STK17A could help in identifying earlier changes during the evolution of chronic rejection (CR) after liver transplantation. RT-PCR and immunofluorescence were used to analyze STK17A expression in HepG2 cells. Antibody microarray was performed using human liver samples from CR and healthy donors. Immunohistochemistry was used to verify the clinical utility of STK17A on sequential biopsies for the subsequent development of CR. A novel short isoform of STK17A was found in HepG2 cells. STK17A was localized in the nuclei and bile canaliculi in HepG2 cells and human livers. Microarray of STK17A revealed its decrease in failed liver allografts by CR. During the evolution of CR, the staining pattern of bile canalicular STK17A gradually changed from diffuse linear to focal intermittent. The focal intermittent staining pattern was observed before the definite diagnosis of CR. In conclusion, the present study was the first to find localization of STK17A in normal bile canaliculi. Abnormal expression and localization of STK17A were associated with CR of liver allografts since the early stage of the rejection process.     

Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space

Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions.     

LC3B is indispensable for selective autophagy of p62 but not basal autophagy

Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs.     

Ubiquitin ligase CHIP suppresses cancer stem cell properties in a population of breast cancer cells

Cancer stem cells (CSCs) have several distinctive characteristics, including high metastatic potential, tumor-initiating potential, and properties that resemble normal stem cells such as self-renewal, differentiation, and drug efflux. Because of these characteristics, CSC is regarded to be responsible for cancer progression and patient prognosis. In our previous study, we showed that a ubiquitin E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) suppressed breast cancer malignancy. Moreover, a recent clinical study reported that CHIP expression levels were associated with favorable prognostic parameters of patients with breast cancer. Here we show that CHIP suppresses CSC properties in a population of breast cancer cells. CHIP depletion resulted in an increased proportion of CSCs among breast cancers when using several assays to assess CSC properties. From our results, we propose that inhibition of CSC properties may be one of the functions of CHIP as a suppressor of cancer progression.