Redox regulation of the protein tyrosine phosphatase PTP1B in cancer cells

The oxidation and inactivation of protein tyrosine phosphatases is one mechanism by which reactive oxygen species influence tyrosine phosphorylation-dependent signaling events and exert their biological functions. In the present study, we determined the redox status of endogenous protein tyrosine phosphatases in HepG2 and A431 human cancer cells, in which reactive oxygen species are produced constitutively. We used mass spectrometry to assess the state of oxidation of the catalytic cysteine residue of endogenous PTP1B and show that this residue underwent both reversible and irreversible oxidation to high stoichiometry in response to intrinsic reactive oxygen species production. In addition, our data show that the oxidation of PTP1B is specific to the active site Cys, with the other Cys residues in the protein remaining in a reduced state. Treatment of these cells with diphenyleniodonium, an inhibitor of NADPH oxidases, decreased reactive oxygen species levels. This resulted in inhibition of protein tyrosine phosphatase oxidation, concomitant with decreased tyrosine phosphorylation of cellular proteins and inhibition of anchorage-independent cell growth. Therefore, our data also suggest that the high level of intrinsic reactive oxygen species may contribute to the transformed phenotype of HepG2 and A431 cells via constitutive inactivation of cellular protein tyrosine phosphatases.     

Occurrence of arbuscular mycorrhiza in Castanospermum australe A. Cunn. & C. Fraser and effects on growth and production of castanospermine

 Castanospermum australe A. Cunn. & C. Fraser is the only species of the genus Castanospermum (the Moreton Bay chestnut or black bean) native to NE Australia. One constituent of the plant, castanospermine, can inhibit the AIDS virus. The present study investigated possible symbioses between its roots and arbuscular mycorrhizal (AM) fungi. The effects of mycorrhizal fungi on the growth of the plant and yield of alkaloid castanospermine were also studied. The mycorrhizosphere soil and roots of C. australe collected from various sites in and around Sydney, Australia showed AM symbiotic associations with roots, with arbuscules and vesicles in the root cortices. Wet sieving and decanting yielded AM fungal spores, mainly Glomus spp. A positive correlation was found between AM fungal infection and the castanospermine content of seeds of field-grown trees. Field study results were confirmed by growing seedlings under greenhouse conditions and inoculating them with Glomus intraradices Schenck and Smith (INVAM isolate KS906) and Gigaspora margarita Becker & Hall (INVAM isolate BR444–2). The AM fungi increased the growth and P contents of plants and the yield of castanospermine in the leaves, irrespective of the P treatment. No correlation was found between the alkaloid contents of leaves from mycorrhizal seedlings and from non-mycorrhizal plants which received P. No significant difference in the production of castanospermine was found between P treatments when G. margarita was used as inoculum. Accepted: 14 April 1999     

Adenosine induced apoptosis in BHK cells via P1 receptors and equilibrative nucleoside transporters

Adenosine, as a ubiquitous metabolite, mediates many physiological functions via activation of plasma membrane receptors. Mechanisms of most of its physiological roles have been studied extensively, but research on adenosine-induced apoptosis (AIA) has only started recently. In this study we demonstrate that adenosine dose-dependently triggered apoptosis of cultured baby hamster kidney (BHK) cells. Adenosine-induced apoptotic cell death was characterized by DNA laddering, changes in nuclear chromatin morphology and phosphatidylserine staining. Apoptosis was also quantified by flow cytometry. Results suggest the involvement of adenosine A1 and A3 receptors as well as equilibrative nucleoside transporters in apoptosis induced by adenosine. These results indicate a receptor-transporter co-signaling mechanism in AIA in BHK cells. The involvement of A1 and A3 receptors also implies a possible apoptotic pathway mediated by G protein-coupled receptors.     

Genetic Diversity Within and Among Feral Populations and Domesticated Strains of the Guppy (Poecilia reticulata) in Singapore

Genetic variability within and among feral populations and cultured strains of the guppy (Poecilia reticulata) was investigated by random amplification of polymorphic DNA (RAPD) fingerprinting. Feral guppies were collected from 6 isolated populations (BT, Bukit Timah; NS, Nee Soon; TS, Tuas; MF, Mount Faber; KR, Kranji; LI, laboratory-inbred feral line), while the Tuxedo and Green Variegated strains were sampled from 2 guppy farms in Singapore. Pairwise genetic distances analyzed by unweighted pair-group method with arithmetic means revealed distinct clustering of guppy individuals into their respective populations and strains. Percentage polymorphic loci ranged from 54.96% (TS) to 68.70% (KR), while average heterozygosity ranged from 0.220 (GV) to 0.271 (KR). In contrast, TS guppies had the highest (0.850) intrapopulation genetic similarity (S), whereas KR had the lowest (0.781). Among populations and strains, S ranged from 0.703 (between GV and LI) to 0.809 (between NS and MB). The GV strain S was closer to TX (0.784) than to the feral guppies. Bootstrapped genetic distance trees depicted 3 major nodes comprising BT-TS, NS-MF, and TX-GV. Principal coordinate analysis also differentiated the 6 feral populations from the 2 cultured strains.     

Pten dose dictates cancer progression in the prostate

Complete inactivation of the PTEN tumor suppressor gene is extremely common in advanced cancer, including prostate cancer (CaP). However, one PTEN allele is already lost in the vast majority of CaPs at presentation. To determine the consequence of PTEN dose variations on cancer progression, we have generated by homologous recombination a hypomorphic Pten mouse mutant series with decreasing Pten activity: Pten^hy/+ > Pten^+/− > Pten^hy/− (mutants in which we have rescued the embryonic lethality due to complete Pten inactivation) > Pten prostate conditional knockout (Pten^pc) mutants. In addition, we have generated and comparatively analyzed two distinct Pten^pc mutants in which Pten is inactivated focally or throughout the entire prostatic epithelium. We find that the extent of Pten inactivation dictate in an exquisite dose-dependent fashion CaP progression, its incidence, latency, and biology. The dose of Pten affects key downstream targets such as Akt, p27^Kip1, mTOR, and FOXO3. Our results provide conclusive genetic support for the notion that PTEN is haploinsufficient in tumor suppression and that its dose is a key determinant in cancer progression.     

Growth and differentiation of embryoid bodies derived from human embryonic stem cells: effect of glucose and basic fibroblast growth factor

Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). This differentiation occurs following suspension culturing of EBs in media containing a high (25 mM) glucose concentration. Although high-glucose-containing media is used for maintenance and proliferation of ES cells, it has not been demonstrated whether this is a necessary requirement for EB development. To address this, we examined the growth and differentiation of EBs established in 0-mM, 5.5-mM (physiological), and 25-mM (high) glucose concentrations, through morphometric analysis and examination of gene and protein expression. The effect on EB development of supplementation with basic fibroblast growth factor (FGF2) was also studied. We report that the greatest rate of EB growth occurs in 5.5 mM glucose media. A morphological study of EBs over 104 days duration under glucose-containing conditions demonstrated the development of all three major embryonic cell types. The difference from normal human development was obvious in the lack of rostrocaudal control by the notochord. In the latest stages of development, the main tissue observed appeared to be cartilage and cells of a mesodermal lineage. We conclude that physiological glucose concentrations are suitable for the culturing of EBs, that the addition of FGF2 enhances the temporal expression of genes including POU5F1, nestin, FOXA2, ONECUT1, NEUROD1, PAX6, and insulin, and that EBs can be cultured in vitro for long periods, allowing for further examination of developmental processes.     

Regionalization of cell fates and cell movement in the endoderm of the mouse gastrula and the impact of loss of Lhx1(Lim1) function

Investigation of the developmental fates of cells in the endodermal layer of the early bud stage mouse embryo revealed a regionalized pattern of distribution of the progenitor cells of the yolk sac endoderm and the embryonic gut. By tracing the site of origin of cells that are allocated to specific regions of the embryonic gut, it was found that by late gastrulation, the respective endodermal progenitors are already spatially organized in anticipation of the prospective mediolateral and anterior-posterior destinations. The fate-mapping data further showed that the endoderm in the embryonic compartment of the early bud stage gastrula still contains cells that will colonize the anterior and lateral parts of the extraembryonic yolk sac. In the Lhx1(Lim1)-null mutant embryo, the progenitors of the embryonic gut are confined to the posterior part of the endoderm. In particular, the prospective anterior endoderm was sequestered to a much smaller distal domain, suggesting that there may be fewer progenitor cells for the anterior gut that is poorly formed in the mutant embryo. The deficiency of gut endoderm is not caused by any restriction in endodermal potency of the mutant epiblast cells but more likely the inadequate allocation of the definitive endoderm. The inefficient movement of the anterior endoderm, and the abnormal differentiation highlighted by the lack of Sox17 and Foxa2 expression, may underpin the malformation of the head of Lhx1 mutant embryos.     

Optical immunoassay for snake venom detection

A sensitive and specific optical immunoassay (OIA) has been developed for snake venom detection. The assay is based on the principle of detection of physical changes in thickness of molecular thin film resulting from specific binding events on an optical silicon chip (SILAS-I, ThermoBioStar, Colorado, USA). The reflection of white light through the thin film results in destructive interference of a particular wavelength of the light from gold to purple-blue depending on the thickness of the thin film formed or the amount of venom in the test sample. A prototype test kit for the simultaneous identification of species and semi-quantitative detection of venoms from four medically important snakes of South Vietnam (Trimeresurus albolabris, Calloselasma rhodostoma, Naja kaouthia and Ophiophagus hannah) has been developed. The kit can detect venom analytes in blood, plasma, urine, wound exudates, blister fluid or tissue homogenates. The efficacy of the test kit in snakebite diagnosis has been demonstrated in experimental envenomations and sample analytes taken from snakebite victims in South Vietnam. This rapid snake venom detection kit based on OIA technique is potentially applicable in the clinics as well as in the field.