Modeling breast cancer in vivo and ex vivo reveals an essential role of Pin1 in tumorigenesis

Phosphorylation on certain Ser/Thr-Pro motifs is a major oncogenic mechanism. The conformation and function of phosphorylated Ser/Thr-Pro motifs are further regulated by the prolyl isomerase Pin1. Pin1 is prevalently overexpressed in human cancers and implicated in oncogenesis. However, the role of Pin1 in oncogenesis in vivo is not known. We have shown that Pin1 ablation is highly effective in preventing oncogenic Neu or Ras from inducing cyclin D1 and breast cancer in mice, although it neither affects transgene expression nor mammary gland development. Moreover, we have developed an ex vivo assay to uncover that a significant fraction of primary mammary epithelial cells from Neu or Ras mice display various malignant properties long before they develop tumors in vivo. Importantly, these early transformed properties are effectively suppressed by Pin1 deletion, which can be fully rescued by overexpression of cyclin D1. Thus, Pin1 is essential for tumorigenesis and is an attractive anticancer target. Our ex vivo assay can be used to study early events of breast cancer development in genetically predisposed mice.     

Isolation and characterization of the tobramycin biosynthetic gene cluster from Streptomyces tenebrarius

The biosynthetic gene cluster for tobramycin, a 2-deoxystreptamine-containing aminoglycoside antibiotic, was isolated from Streptomyces tenebrarius ATCC 17920. A genomic library of S. tenebrarius was constructed, and a cosmid, pST51, was isolated by the probes based on the core regions of 2-deoxy-scyllo-inosose (DOI) synthase, and L-glutamine:DOI aminotransferase and L-glutamine:scyllo-inosose aminotransferase. Sequencing of 33.9 kb revealed 24 open reading frames (ORFs) including putative tobramycin biosynthetic genes. We demonstrated that one of these ORFs, tbmA, encodes DOI synthase by in vitro enzyme assay of the purified protein. The catalytic residues of TbmA and dehydroquinate synthase were studied by homology modeling. The gene cluster found is likely to be involved in the biosynthesis of tobramycin.     

Overexpression of OsRAA1 causes pleiotropic phenotypes in transgenic rice plants, including altered leaf, flower, and root development and root response to gravity

There are very few root genes that have been described in rice as a monocotyledonous model plant so far. Here, the OsRAA1 (Oryza sativa Root Architecture Associated 1) gene has been characterized molecularly. OsRAA1 encodes a 12.0-kD protein that has 58% homology to the AtFPF1 (Flowering Promoting Factor 1) in Arabidopsis, which has not been reported as modulating root development yet. Data of in situ hybridization and OsRAA1::GUS transgenic plant showed that OsRAA1 expressed specifically in the apical meristem, the elongation zone of root tip, steles of the branch zone, and the young lateral root. Constitutive expression of OsRAA1 under the control of maize (Zea mays) ubiquitin promoter resulted in phenotypes of reduced growth of primary root, increased number of adventitious roots and helix primary root, and delayed gravitropic response of roots in seedlings of rice (Oryza sativa), which are similar to the phenotypes of the wild-type plant treated with auxin. With overexpression of OsRAA1, initiation and growth of adventitious root were more sensitive to treatment of auxin than those of the control plants, while their responses to 9-hydroxyfluorene-9-carboxylic acid in both transgenic line and wild type showed similar results. OsRAA1 constitutive expression also caused longer leaves and sterile florets at the last stage of plant development. Analysis of northern blot and GUS activity staining of OsRAA1::GUS transgenic plants demonstrated that the OsRAA1 expression was induced by auxin. At the same time, overexpression of OsRAA1 also caused endogenous indole-3-acetic acid to increase. These data suggested that OsRAA1 as a new gene functions in the development of rice root systems, which are mediated by auxin. A positive feedback regulation mechanism of OsRAA1 to indole-3-acetic acid metabolism may be involved in rice root development in nature.     

The progressive changes of nasal symmetry and growth after nasoalveolar molding: a three-year follow-up study

The purpose of this study was to assess the progressive changes of nasal symmetry, growth, and relapse after presurgical nasoalveolar molding and primary cheiloplasty in unilateral complete cleft lip/palate infants. Twenty-five consecutive complete unilateral cleft lip/palate infants were included. All the infants underwent nasoalveolar molding before primary cheiloplasty. Standard 1:1 ratio basilar photographs were taken before and after nasoalveolar molding, 1 week after cheiloplasty, and yearly for 3 years. Linear measurements were made directly on the photographs. The results of this study revealed that the nasal asymmetry was significantly improved after nasoalveolar molding and was further corrected to symmetry after primary cheiloplasty. After the primary cheiloplasty, the nasal asymmetry significantly relapsed in the first year postoperatively and then remained stable and well afterward. The relapse was the result of a significant differential growth between the cleft and noncleft sides in the first year postoperatively. To compensate for relapse and differential growth, the authors recommend (1) narrowing down the alveolar cleft as well as possible by nasoalveolar molding, (2) overcorrecting the nasal vertical dimension surgically, and (3) maintaining the surgical results using a nasal conformer.     

Intra-aneurysmal flow with helix and mesh stent placement across side-wall aneurysm pore of a straight parent vessel

Pulsatile flow fields in a cerebrovascular side-wall aneurysm model with a wide ostium after stenting are presented in terms of particle tracking velocimetry measurements and flow visualization. Among the stent parameters the shape, helix versus mesh, was selected to study its effect on the changes of intraaneurysmal hemodynamics for the reference of minimally invasive endovascular aneurysm treatment. The blocking ratio of the stents was fixed at 30%. The Womersley number was 3.9 and the mean, peak, and minimal Reynolds numbers based on the bulk average velocity and diameter of the parent vessel were 600, 850, and 300, respectively. Four consecutive flow-rate phases were selected to characterize the intra-aneurysmal flow. The results are characterized in terms of velocity vector field, regional average velocity, and intra-aneurysmal vorticity/circulation/wall shear stress. It is found that the hemodynamic features inside the aneurysm alter markedly with the shape of the stent and the size of the orifice. Both stents investigated induce favorable changes in the intra-aneurysmal flow stasis as well as direction and undulation of wall shear stresses. A comparison of the results of the helix to mesh stent shows that the former is more favorable for endovascular treatment.     

DNA-induced aggregation of zwitterionic oligolamellar liposome

Liposome consisting of a single zwitterionic lipid as the potential vector for gene therapy has been reported recently; however, whether polyanionic DNA can bind directly with zwitterionic lipid without the aid of multivalent salt still remains unresolved. In this study, we reveal the aggregation of zwitterionic oligolamellar liposomes composed of 1,2-di(cis-9-octadecenoyl)-sn-glycero-3-phosphocholine induced by DNA without the presence of multivalent salt. Our results demonstrate that only a small fraction (<10%) of DNA can bind electrostatically with a portion of the liposomes. Such a low degree of binding, however, induces significant aggregation of these oligolamellar liposomes, yielding large multilamellar particles in which the number of hydrophilic/hydrophobic layer stacking becomes sufficiently large to yield multiple diffraction peaks in the small-angle X-ray scattering profile. Addition of monovalent salt such as NaCl tends to disrupt the multilamellar structure.     

One-pot enzymatic production of dTDP-4-keto-6-deoxy-D-glucose from dTMP and glucose-1-phosphate

An enzymatic production method for dTDP-4-keto-6-deoxy-D-glucose, a key intermediate of various deoxysugars in antibiotics, was developed starting from dTMP, acetyl phosphate, and glucose-1-phosphate. Four enzymes, i.e., TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-D-glucose 4,6-dehydratase' were overexpressed using T7 promoter system in the E. coli BL21 strain, and the dTDP-4-keto-6-deoxy-D-glucose was synthesized by using the enzyme extracts in one-pot batch system. When 20 mM dTMP of initial concentration was used, Mg2+ ion, acetyl phosphate, and glucose-1-phosphate concentrations were optimized. About 95% conversion yield of dTDP-4-keto-6-deoxy-D-glucose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 60 mM acetyl phosphate, 80 mM glucose-1-phosphate, and 20 mM MgCl(2). The rate-limiting step in this multiple enzyme reaction system was the dTDP-glucose synthase reaction. Using the reaction scheme, about 1 gram of purified dTDP-4-keto-6-deoxy-D-glucose was obtained in an overall yield of 81% after two-step purification, i.e., anion exchange chromatography and gel filtration.     

Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18

We present the 4.8-Mb complete genome sequence of Salmonella enterica serovar Typhi strain Ty2, a human-specific pathogen causing typhoid fever. A comparison with the genome sequence of recently isolated S. enterica serovar Typhi strain CT18 showed that 29 of the 4,646 predicted genes in Ty2 are unique to this strain, while 84 genes are unique to CT18. Both genomes contain more than 200 pseudogenes; 9 of these genes in CT18 are intact in Ty2, while 11 intact CT18 genes are pseudogenes in Ty2. A half-genome interreplichore inversion in Ty2 relative to CT18 was confirmed. The two strains exhibit differences in prophages, insertion sequences, and island structures. While CT18 carries two plasmids, one conferring multiple drug resistance, Ty2 has no plasmids and is sensitive to antibiotics.     

Microarray profiling of gene expression patterns in bladder tumor cells treated with genistein

Microarray technology was used to gain an insight into the molecular events of tumor cell growth inhibition mediated by the soy isoflavone genistein. For this, a susceptible bladder tumor line TCCSUP was treated with the inhibitory dose (50 microM) of genistein for various periods of time, followed by mRNA isolations, cDNA probe preparations, and hybridization individually to cDNA chips containing 884 sequence-verified known human genes. After analyzing the hybridization signals with a simple quantitative method developed by this study, we detected that egr-1, whose expression has been associated with proliferation and differentiation, was transiently induced and this expression pattern was later confirmed by RT-PCR. Thus, microarray technology is a reliable and powerful tool for profiling gene expression patterns in many biological systems related to cancer. We further detected many groups of genes with distinct expression profiles and most of them encode for proteins that regulate the signal transduction or the cell cycle pathways. These genes warrant further investigation as regards their roles in the susceptibility of the tumor cell line to the antitumor drug.