Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible genes in human amniotic epithelial cells

Background  

Exposure to dioxins results in a broad range of pathophysiological disorders in human fetuses. In order to evaluate the effects of dioxins on the feto-placental tissues, we analyzed the gene expression in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated primary cultures of human amniotic epithelial cells.     

A straightforward protocol for the preparation of high performance microarray displaying synthetic MUC1 glycopeptides

Background

Human serum MUC1 peptide fragments bearing aberrant O-glycans are secreted from columnar epithelial cell surfaces and known as clinically important serum biomarkers for the epithelial carcinoma when a specific monoclonal antibody can probe disease-relevant epitopes. Despite the growing importance of MUC1 glycopeptides as biomarkers, the precise epitopes of most anti-MUC1 monoclonal antibodies remains unclear.

Methods

A novel protocol for the fabrication of versatile microarray displaying peptide/glycopeptide library was investigated for the construction of highly sensitive and accurate epitope mapping assay of various anti-MUC1 antibodies.

Results

Selective imine-coupling between aminooxy-functionalized methacrylic copolymer with phosphorylcholine unit and synthetic MUC1 glycopeptides-capped by a ketone linker at N-terminus provided a facile and seamless protocol for the preparation of glycopeptides microarray platform. It was demonstrated that anti-KL-6 monoclonal antibody shows an extremely specific and strong binding affinity toward MUC1 fragments carrying sialyl T antigen (Neu5Acα2,3Galβ1,3GalNAcα1→) at Pro-Asp-Thr-Arg motif when compared with other seven anti-MUC1 monoclonal antibodies such as VU-3D1, VU-12E1, VU-11E2, Ma552, VU-3C6, SM3, and DF3. The present microarray also uncovered the occurrence of IgG autoantibodies in healthy human sera that bind specifically with sialyl T antigen attached at five potential O-glycosylation sites of MUC1 tandem repeats.

Conclusion

We established a straightforward strategy toward the standardized microarray platform allowing highly sensitive and accurate epitope mapping analysis by reducing the background noise due to nonspecific protein adsorption.

General significance

The present approach would greatly accelerate the discovery research of new class autoantibodies as well as the development of therapeutic mAbs reacting specifically with disease-relevant epitopes.     

Ganglioside GD3 Enhances Adhesion Signals and Augments Malignant Properties of Melanoma Cells by Recruiting Integrins to Glycolipid-enriched Microdomains

Ganglioside GD3 is widely expressed in human malignant melanoma cell lines and tumors. Previously, we reported that GD3+ cells show stronger tyrosine phosphorylation of focal adhesion kinase (FAK), p130^Cas, and paxillin when treated with fetal calf serum than GD3− cells. In this study, we analyzed the changes in the signals mediated by the interaction between integrins and extracellular matrices (ECM) to clarify how GD3 enhances cell signals in the vicinity of the cell membrane. An adhesion assay with a real time cell electronic sensing system revealed that GD3+ cells had stronger adhesion to all extracellular matrices examined. In particular, GD3+ cells attached more strongly to collagen type I and type IV than controls. Correspondingly, they showed stronger tyrosine phosphorylation of FAK and paxillin during adhesion to collagen type I. In the floating pattern of detergent extracts, a high level of integrin β1 was found in glycolipid-enriched microdomain (GEM)/rafts in GD3+ cells before adhesion, whereas a smaller amount of integrin β1 was detected in the GEM/rafts of controls. Some phosphorylated forms of FAK as well as total FAK were found in GEM/rafts during cell adhesion only in GD3+ cells. Another signal consisting of integrin-linked kinase/Akt was also activated during adhesion more strongly in GD3+ cells than in controls. In double stained GD3+ cells, GD3 and integrin β1 co-localized at the focal adhesion with a punctate pattern. All these results suggested that integrins assembled and formed a cluster in GEM/rafts, leading to the enhanced signaling and malignant properties under GD3 expression.     

Suppression of Adipocyte Differentiation by Aldo-keto Reductase 1B3 Acting as Prostaglandin F2�� Synthase

Prostaglandin (PG) F_2α suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor γ. However, PGF_2α synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF_2α, was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF_2α production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF_2α. These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF_2α suppressed adipocyte differentiation by acting through FP receptors.     

Histochemical studies on the morphology of the Golgi apparatus in the neurons of supraoptic and paraventricular nuclei of the normal and dehydrated rabbit (application of the thiamine pyrophosphatase method)

Summary Detailed histochemical studies have been conducted on the morphology of the Golgi apparatus by applying the thiamine pyrophosphatase technique (Novikoff and Goldfisher, 1961) to the neurons of supraoptic and paraventricular nuclei of normal and dehydrated rabbits. The neurons in both nuclei were classified into five categories on the basis of the morphology of the Golgi apparatus. The number of cells in individual categories were counted to evaluate the percentage of each category in the whole nucleus.Neurons have many vesicles which show the tendency to form clusters. Such clusters are present also in the basal bodies. The Golgi apparatus is localized near one side of the nucleus in many neurons. The neurons indicate phasic activity of resting, anabolic and catabolic stages under normal conditions.During dehydration, the Golgi apparatus went through the three stages of network formation, the increase of the budding-off process and later on disintegration. The supraoptic nucleus reacted to the TPPase test more severely than the paraventricular nucleus, whereas the former went through the stages more slowly than the latter. The paraventricular nucleus also revealed sensitivity to osmotic stress.     

Development and characterisation of microsatellite loci in Farfugium japonicum (Asteraceae)

Eight microsatellite loci for the perennial herb Farfugium japonicum, including the rheophytic variety luchuense endemic to riparian areas of the Ryukyu Islands, Japan, were isolated and characterised. The number of alleles ranged from 5 to 14. The expected (H _E) and observed (H _O) heterozygosities were 0.344–0.885 and 0.121–0.754, respectively, from 69 individuals in one population. Six loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium (P < 0.05). The primers amplifying microsatellite sequences in F. japonicum may provide a population genetics tool useful in the establishment of a conservation strategy.     

Microsatellite polymorphism in the human heme oxygenase-1 gene promoter and its application in association studies with Alzheimer and Parkinson disease

Oxidative stress has been suggested to be involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer disease (AD) and Parkinson disease (PD). Heme oxygenase-1 (HO-1), a key enzyme in heme catabolism, also functions as an antioxidant enzyme. Here, we show that a (GT)_n repeat in the human HO-1 gene promoter region is highly polymorphic, although no particular alleles are associated with AD or PD. This newly identified genetic marker should allow us to study the possible involvement of HO-1 in certain human diseases. Received: 5 November 1996 / Accepted: 18 February 1997     

Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli

Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency. A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline. This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein. Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin. However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene. The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes. It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of Guamyl-tRNA for porphyrin synthesis. Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.     

Evaluation of colors in green mutants isolated from purple bacteria as a host for colorimetric whole-cell biosensors

The change in carotenoid-based bacterial color from yellow to red can be applied to whole-cell biosensors. We generated several green mutants to emphasize the color change in such biosensors. The blue-green crtI-deleted mutant, Rhodopseudomonas palustris no.711, accumulated the colorless carotenoid precursor, phytoene. Green Rhodovulum sulfidophilum M31 accumulated neurosporene, a downstream product of phytoene. Another green mutant, Rhodobacter sphaeroides Ga, accumulated neurosporene and chloroxanthin, which are both downstream products of phytoene. All green mutants accumulated bacteriochlorophyll a. Photosynthetic membrane obtained from the green mutants all exhibited decreased absorption of wavelength range at 510–570 nm. Therefore, these indicate that the greenish bacterial colors were mainly caused by the existence of bacteriochlorophyll a and the changes in carotenoid composition in photosynthetic membrane. The colors of the green mutants and their wild-type strains were plotted in the CIE-L*a*b* color space, and the color difference (ΔE*ab) values between a green mutant and its wild type were calculated. ΔE*ab values were higher in the green mutants than in Rdv. sulfidophilum CDM2, the yellowish host strain of reported biosensors. These data indicate that change in bacterial color from green to red is more distinguishable than that from yellow to red as a reporter signal of carotenoid-based whole-cell biosensors.     

Stable integration and conditional expression of electroporated transgenes in chicken embryos

The in ovo electroporation in chicken embryos has widely been used as a powerful tool to study roles of genes during embryogenesis. However, the conventional electroporation technique fails to retain the expression of transgenes for more than several days because transgenes are not integrated into the genome. To overcome this shortcoming, we have developed a transposon-mediated gene transfer, a novel technique in chicken manipulations. It was previously reported that the transposon Tol2, originally found in medaka fish, facilitates an integration of a transgene into the genome when co-acting with Tol2 transposase. In this study, we co-electroporated a plasmid containing a CAGGS-EGFP cassette cloned in the Tol2 construct along with a transposase-encoding plasmid into early presomitic mesoderm or optic vesicles of chicken embryos. This resulted in persistent expression of EGFP at least until embryonic day 8 (E8) and E12 in somite-derived tissues and developing retina, respectively. The integration of the transgene was confirmed by genomic Southern blotting using chicken cultured cells. We further combined this transposon-mediated gene transfer with the tetracycline-dependent conditional expression system that we also developed recently. With this combined method, expression of a stably integrated transgene could be experimentally induced upon tetracycline administration at relatively late stages such as E6, where a variety of organogenesis are underway. Thus, the techniques proposed in this study provide a novel approach to study the mechanisms of late organogenesis, for which chickens are most suitable model animals.