Functional complementation between a novel mammalian polygenic transport complex and an evolutionarily ancient organic solute transporter,OSTalpha-OSTbeta

These studies identify an organic solute transporter (OST) that is generated when two novel gene products are co-expressed, namely human OSTalpha and OSTbeta or mouse OSTalpha and OSTbeta. The results also demonstrate that the mammalian proteins are functionally complemented by evolutionarily divergent Ostalpha-Ostbeta proteins recently identified in the little skate, Raja erinacea, even though the latter exhibit only 25-41% predicted amino acid identity with the mammalian proteins. Human, mouse, and skate OSTalpha proteins are predicted to contain seven transmembrane helices, whereas the OSTbeta sequences are predicted to have a single transmembrane helix. Human OSTalpha-OSTbeta and mouse Ostalpha-Ostbeta cDNAs were cloned from liver mRNA, sequenced, expressed in Xenopus laevis oocytes, and tested for their ability to functionally complement the corresponding skate proteins by measuring transport of [3H]estrone 3-sulfate. None of the proteins elicited a transport signal when expressed individually in oocytes; however, all nine OSTalpha-OSTbeta combinations (i.e. OSTalpha-OSTbeta pairs from human, mouse, or skate) generated robust estrone 3-sulfate transport activity. Transport was sodium-independent, saturable, and inhibited by other steroids and anionic drugs. Human and mouse OSTalpha-OSTbeta also were able to mediate transport of taurocholate, digoxin, and prostaglandin E2 but not of estradiol 17beta-d-glucuronide or p-aminohippurate. OSTalpha and OSTbeta were able to reach the oocyte plasma membrane when expressed either individually or in pairs, indicating that co-expression is not required for proper membrane targeting. Interestingly, OSTalpha and OSTbeta mRNAs were highly expressed and widely distributed in human tissues, with the highest levels occurring in the testis, colon, liver, small intestine, kidney, ovary, and adrenal gland.     

Shear stress activates Tie2 receptor tyrosine kinase in human endothelial cells

The receptor tyrosine kinase (RTK) Tie2 is expressed predominantly on endothelial cells. Tie2 is critical for vasculogenesis during development and could be important for maintaining endothelial cell survival and integrity in adult blood vessels. Although most RTKs are activated by shear stress in the absence of ligand activation, the effect of shear stress on Tie2 is unknown. Therefore, we examined the effect of shear stress on Tie2 phosphorylation in primary cultured endothelial cells. Interestingly, shear stress (20 dyne/cm(2)) produced a rapid, marked, and sustained Tie2 phosphorylation, while it produced a rapid but slight and transient phosphorylation of insulin receptor and VEGF receptor 2 (Flk1). In addition, Tie2 phosphorylation in response to shear stress was velocity-dependent, while phosphorylation of insulin receptor and Flk1 was not. Shear stress also produced Akt phosphorylation in a time-, velocity-, and PI 3-kinase-dependent manner. Accordingly, shear stress suppressed serum deprivation-induced endothelial cell apoptosis. Taken together, our results indicated that activation of Tie2/PI 3-kinase/Akt in response to shear stress could be an important signaling cascade for maintaining endothelial survival and integrity in blood vessels.     

Chromosomal polymorphisms due to heterochromatin growth and pericentric inversions in white-bellied rat,Niviventer confucianus,from China

Different cytogenetic techniques were used to analyze the chromosomes of white-bellied rat, Niviventer confucianus from Mt. Tai and Jinan, Shandong Province and Ningshan, Shaanxi Province of China. Shandong populations have 2n = 46 chromosomes with 4 metacentric, 2 subtelocentric, 16 telocentric pairs of autosomes and the submetacentric X and telocentric Y. The chromosomal arm number (NF) of the two populations was 56. Shaanxi population has 2n = 46 chromosomes with 4 metacentric, 1 submetacentric, 1 subtelocentric and 16 telocentric pairs of autosomes and the submetacentric X and telocentric Y. The karyotype of Ningshan population showed NF = 58. As the result of the comparison of C- and G-banding patterns, and compare with other species in the genus Niviventer, we suppose that the chromosomal evolution of Niviventer involved in pericentric inversion and heterochromatin growth. The submetacentric chromosomes of Shaanxi population would be originated from the growth of heterochromatin of the subtelocentric chromosome of Shandong population.     

Parathyroid hormone and parathyroid hormone-related protein exert both pro- and anti-apoptotic effects in mesenchymal cells

During bone formation, multipotential mesenchymal cells proliferate and differentiate into osteoblasts, and subsequently many die because of apoptosis. Evidence suggests that the receptor for parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), the PTH-1 receptor (PTH-1R), plays an important role in this process. Multipotential mesenchymal cells (C3H10T1/2) transfected with normal or mutant PTH-1Rs and MC3T3-E1 osteoblastic cells were used to explore the roles of PTH, PTHrP, and the PTH-1R in cell viability relative to osteoblastic differentiation. Overexpression of wild-type PTH-1R increased cell numbers and promoted osteocalcin gene expression versus inactivated mutant receptors. Furthermore, the effects of PTH and PTHrP on apoptosis were dramatically dependent on cell status. In preconfluent C3H10T1/2 and MC3T3-E1 cells, PTH and PTHrP protected against dexamethasone-induced reduction in cell viability, which was dependent on cAMP activation. Conversely, PTH and PTHrP resulted in reduced cell viability in postconfluent cells, which was also dependent on cAMP activation. Further, the proapoptotic-like effects were associated with an inhibition of Akt phosphorylation. These data suggest that parathyroid hormones accelerate turnover of osteoblasts by promoting cell viability early and promoting cell departure from the differentiation program later in their developmental scheme. Both of these actions occur at least in part via the protein kinase A pathway.     

Inhibitors of NF-kappaB signaling: design and synthesis of a biotinylated isopanepoxydone affinity reagent

A number of inhibitors of NF-kappaB signaling arising from our recent syntheses of isopanepoxydone and panepoxydone have been identified. Structure-activity data have been correlated to allow the design and synthesis of an affinity reagent for the isolation and identification of any relevant cellular target.     

Cytologic diagnosis of estrogen and progesterone receptors in breast imprints

OBJECTIVE: To investigate estrogen receptor (ER) and progesterone receptor (PR) levels in imprint specimens obtained at breast surgery and to compare their correlation with that of standard methods. STUDY DESIGN: Imprint specimens for cytology were obtained from 101 mass-forming lesions in 66 patients, and specimens were frozen in liquid nitrogen for later assay. The imprint specimens were immunocytochemically (ICC) stained by monoclonal antibody to ER or PR; diaminobenzidine-stained cell nuclei in clusters were regarded as positive. Tissue specimens were assayed by the standard method of dextran-coated charcoal assay (DCC) and enzyme immunoassay. RESULTS: Forty-five primary breast cancer lesions, 2 contralateral breast cancer, 49 dissected nodes and 5 benign breast lesions were collected. The correlation between DCC and ICC was 81% (82/101) for ER and 74% (66/101) for PR. That between EIA and ICC was 88% (88/99) for ER and 80% (79/100) for PR, higher than that between DCC and ICC for ER and PR. CONCLUSION: ICC assessment of ER or PR on imprint cytology is a promising clinical test with an acceptable correlation.