Purification and characterization of protein tyrosine phosphatase PTP-MEG2

PTP-MEG2 is an intracellular protein tyrosine phosphatase with a putative lipid-binding domain at the N-terminus. The present study reports expression, purification, and characterization of the full-length form of the enzyme plus a truncated form containing the catalytic domain alone. Full-length PTP-MEG2 was expressed with an adenovirus system and purified from cytosolic extracts of human 293 cells infected with the recombinant adenovirus. The purification scheme included chromatographic separation of cytosolic extracts on fast flow Q-Sepharose, heparin-agarose, l-histidyldiazobenzylphosphonic acid agarose, and hydroxylapatite. The enrichment of PTP-MEG2 from the cytosol was about 120-fold. The truncated form of PTP-MEG2 was expressed in E. coli cells as a non-fusion protein and purified by using a chromatographic procedure similar to that used for the full-length enzyme. The purified full-length and truncated enzymes showed single polypeptide bands on SDS-polyacrylamide gel electrophoresis under reducing conditions and behaved as monomers on gel exclusion chromatography. With para-nitrophenylphosphate and phosphotyrosine as substrates, both forms of the enzyme exhibited classical Michaelis-Menten kinetics. Their responses to pH, ionic strength, metal ions, and protein phosphatase inhibitors are similar to those observed with other characterized tyrosine phosphatases. Compared with full-length PTP-MEG2, the truncated DeltaPTP-MEG2 displayed significantly higher V(max) and lower K(m) values, suggesting that the N-terminal putative lipid-binding domain may have an inhibitory role. The full-length and truncated forms of PTP-MEG2 were also expressed as GST fusion proteins in E. coli cells and purified to near homogeneity through affinity columns. However, the specific phosphatase activities of the GST fusion proteins were 10-25-fold below those obtained with the correspondent non-fusion proteins.     

Pharmacogenetic evidence that cd36 is a key determinant of the metabolic effects of pioglitazone

Pioglitazone, like other thiazolidinediones, is an insulin-sensitizing agent that activates the peroxisome proliferator-activated receptor gamma and influences the expression of multiple genes involved in carbohydrate and lipid metabolism. However, it is unknown which of these many target genes play primary roles in determining the antidiabetic and hypolipidemic effects of thiazolidinediones. To specifically investigate the role of the Cd36 fatty acid transporter gene in the insulin-sensitizing actions of thiazolidinediones, we studied the metabolic effects of pioglitazone in spontaneously hypertensive rats (SHR) that harbor a deletion mutation in Cd36 in comparison to congenic and transgenic strains of SHR that express wild-type Cd36. In congenic and transgenic SHR with wild-type Cd36, administration of pioglitazone was associated with significantly lower circulating levels of fatty acids, triglycerides, and insulin as well as lower hepatic triglyceride levels and epididymal fat pad weights than in SHR harboring mutant Cd36. Additionally, insulin-stimulated glucose oxidation in isolated soleus muscle was significantly augmented in pioglitazone-fed rats with wild-type Cd36 versus those with mutant Cd36. The Cd36 genotype had no effect on pioglitazone-induced changes in blood pressure. These findings provide direct pharmacogenetic evidence that in the SHR model, Cd36 is a key determinant of the insulin-sensitizing actions of a thiazolidinedione ligand of peroxisome proliferator-activated receptor gamma.     

Endocytosis of epidermal growth factor receptor regulated by Grb2-mediated recruitment of the Rab5 GTPase-activating protein RN-tre

The Grb2 adaptor protein is best known for its role in signaling to the small GTPase p21(ras), mediated through its interaction with the SOS guanine nucleotide exchange factor. Here, we demonstrate that Grb2 also signals to Rab5, a small GTPase that plays a key role in early endocytic trafficking. Grb2 functions through association with RN-tre, a GTPase-activating protein for Rab5. Grb2 and RN-tre associate both in vitro and in vivo, with interaction mediated by both SH3 domains of Grb2 and extended proline-rich sequences in RN-tre. Association between Grb2 and RN-tre is constitutive and occurs independently of Eps8, a previously identified binding partner of RN-tre. Epidermal growth factor (EGF) stimulates recruitment of RN-tre to the EGF receptor (EGFR) in a Grb2-dependent manner. Grb2 and the EGFR are internalized and co-localized in endocytic vesicles in response to EGF. Overexpression of RN-tre blocks the internalization of both proteins, consistent with its function as a negative regulator of Rab5 and endocytosis. Strikingly, RN-tre does not block EGF-induced internalization of a Grb2 mutant deficient in RN-tre binding. These results 1) suggest that the ability of RN-tre to inhibit internalization of the EGFR requires Grb2-mediated binding to the receptor and 2) identify Grb2 as a critical regulator of Rab5 and EGFR endocytosis.     

Beta-carbolines as specific inhibitors of cyclin-dependent kinases

Harmine (3), 7-fluoro-1-methyl beta-carboline (35) and 1-(5-methyl-imidazol-4-yl) beta-carboline (41) were potent and specific inhibitors of cyclin-dependent kinases. The degree of aromaticity of the tricyclic ring and the positioning of substituents are important for inhibitory activity. While most beta-carbolines inhibited CDK2 and CDK5 to the same extent, selective inhibition against CDK2 was observed in 1-(2-chlorophenyl)- (12), 1-(2-fluorophenyl)- (15), and 1-(2-chloro-5-nitrophenyl)- (28) beta-carbolines.     

Molecular and genetic aspects of plant responses to osmotic stress

Drought, high salinity and freezing impose osmotic stress on plants. Plants respond to the stress in part by modulating gene expression, which eventually leads to the restoration of cellular homeostasis, detoxification of toxins and recovery of growth. The signal transduction pathways mediating these adaptations can be dissected by combining forward and reverse genetic approaches with molecular, biochemical and physiological studies. Arabidopsis is a useful genetic model system for this purpose and its relatives including the halophyte Thellungiella halophila, can serve as valuable complementary genetic model systems.     

IL-16 regulation of human mast cells/basophils and their susceptibility to HIV-1

AIDS patients often contain HIV-1-infected mast cells (MCs)/basophils in their peripheral blood, and in vivo-differentiated MCs/basophils have been isolated from the blood of asthma patients that are HIV-1 susceptible ex vivo due to their surface expression of CD4 and varied chemokine receptors. Because IL-16 is a ligand for CD4 and/or an undefined CD4-associated protein, the ability of this multifunctional cytokine to regulate the development of human MCs/basophils from nongranulated progenitors residing in cord or peripheral blood was evaluated. After 3 wk of culture in the presence of c-kit ligand, IL-16 induced the progenitors residing in the blood of normal individuals to increase their expression of chymase and tryptase about 20-fold. As assessed immunohistochemically, >80% of these tryptase(+) and/or chymase(+) cells expressed CD4. The resulting cells responded to IL-16 in an in vitro chemotaxis assay, and this biologic response could be blocked by anti-IL-16 and anti-CD4 Abs as well as by a competitive peptide inhibitor corresponding to a sequence in the C-terminal domain of IL-16. The additional finding that IL-16 induces calcium mobilization in the HMC-1 cell line indicates that IL-16 acts directly on MCs and their committed progenitors. IL-16-treated MCs/basophils also are less susceptible to infection by an M/R5-tropic strain of HIV-1. Thus, IL-16 regulates MCs/basophils at a number of levels, including their vulnerability to retroviral infection.     

A cytochrome b origin of photosynthetic reaction centers: an evolutionary link between respiration and photosynthesis

The evolutionary origin of photosynthetic reaction centers has long remained elusive. Here, we use sequence and structural analysis to demonstrate an evolutionary link between the cytochrome b subunit of the cytochrome bc(1) complex and the core polypeptides of the photosynthetic bacterial reaction center. In particular, we have identified an area of significant sequence similarity between a three contiguous membrane-spanning domain of cytochrome b, which contains binding sites for two hemes, and a three contiguous membrane-spanning domain in the photosynthetic reaction center core subunits, which contains binding sites for cofactors such as (bacterio)chlorophylls, (bacterio)pheophytin and a non-heme iron. Three of the four heme ligands in cytochrome b are found to be conserved with the cofactor ligands in the reaction center polypeptides. Since cytochrome b and reaction center polypeptides both bind tetrapyrroles and quinones for electron transfer, the observed sequence, functional and structural similarities can best be explained with the assumption of a common evolutionary origin. Statistical analysis further supports a distant but significant homologous relationship. On the basis of previous evolutionary analyses that established a scenario that respiration evolved prior to photosynthesis, we consider it likely that cytochrome b is the evolutionary precursor for type II reaction center apoproteins. With a structural analysis confirming a common evolutionary origin of both type I and type II reaction centers, we further propose a novel "reaction center apoprotein early" hypothesis to account for the development of photosynthetic reaction center holoproteins.     

Chickens' Cry2: molecular analysis of an avian cryptochrome in retinal and pineal photoreceptors

We have identified and characterized an ortholog of the putative mammalian clock gene cryptochrome 2 (Cry2) in the chicken, Gallus domesticus. Northern blot analysis of gCry2 mRNA indicates widespread distribution in central nervous and peripheral tissues, with very high expression in pineal and retina. In situ hybridization of chick brain and retina reveals expression in photoreceptors and in visual and circadian system structures. Expression is rhythmic; mRNA levels predominate in late subjective night. The present data suggests that gCry2 is a candidate avian clock gene and/or photopigment and set the stage for functional studies of gCry2.     

Physical mapping of the 5S and 45S rDNA in teosintes

The physical locations of the 5S and 45S rDNA sequences were examined in three types of teosinte, Zea mays ssp. mexicana (2n = 20), Zea diploperennis (2n = 20) and Zea perennis (2n = 40) by biotinylated fluorescence in situ hybridization (FISH). The tested materials only showed one hybridization site of 5S rDNA on their genomes, but they were different in the position of the signals. The hybridization site of Zea mays ssp. mexicana was located on the long arm of chromosome 2, indicating that it is the same as the cultivated maize in the position of 5S rDNA, while the sites of Zea diploperennis and Zea perennis were on the short arms of other chromosomes. For 45S rDNA, one hybridization site was detected at secondary constriction region of the satellite chromosomes in Zea mays ssp. mexicana and Zea diploperennis, while in Zea perennis, besides the site located at the secondary constriction region, a second site on the short arm of another chromosome pair was observed. Our results provide additional evidence for Zea mays ssp. mexicana being a subspecies of Zea mays.