Structural Basis for Conformational Dynamics of GTP-bound Ras Protein

Ras family small GTPases assume two interconverting conformations, “inactive” state 1 and “active” state 2, in their GTP-bound forms. Here, to clarify the mechanism of state transition, we have carried out x-ray crystal structure analyses of a series of mutant H-Ras and M-Ras in complex with guanosine 5′-(β,γ-imido)triphosphate (GppNHp), representing various intermediate states of the transition. Crystallization of H-RasT35S-GppNHp enables us to solve the first complete tertiary structure of H-Ras state 1 possessing two surface pockets unseen in the state 2 or H-Ras-GDP structure. Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the ³¹P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions. Collectively, these results clarify the structural features distinguishing the two states and provide new insights into the molecular basis for the state transition of Ras protein.     

Production of daunorubicin by immobilized growing Streptomyces peucetius cells

Summary Streptomyces peucetius cells were immobilized by entrapment in calcium alginate and a photosensitive synthetic polymer, and used for the production of daunorubicin (daunomycin), which is known to be an antitumour reagent. The use of cultivation media removed insoluble components in a natural medium prevented rapid decrease in daunorubicin titer after maximum production. These entrapped cells could be used at least five times for repeated daunorubicin production; the total cultivation period was 45 days.     

Oxygen diffusivity of synthetic gels derived from prepolymers

Summary The oxygen-diffusivity (D _m ) of 16 different gels formed with synthetic prepolymers (photo-crosslinkable resins, urethane resins and photosensitive resins), and that of calcium alginate (for comparison) was determined, using an oxygen electrode covered by the gel membranes with stepwise enzymatic removal of O₂ from the buffer solution. The water content of the gels was found to be decisive for the O₂-diffusivity of the gels: gels with the highest water content showed also the highest D _m . From these findings, the suitability of different polymeric gels for substrate conversion and biosensor systems could be predicted.Abbreviations A surface area of the cathode - c O₂-concentration in the membrane - d _m total thickness of the membrane - D _m O₂-diffusivity in the membrane - ENT, ENTP polymers prepared from hydroxyethylacrylate - ENTA, ENTC isophorone diisocyanate and linear skeleton of different molecular weight of poly(ethylene glycol) (ENT) or poly(propylene glycol) (ENTP), resp. ENTA in addition bears anionic groups, ENTC cationic groups - F Faraday's constant - i _s steady-state O₂ reduction current - N number of electrons per mole unit of reaction - PU polyurethane polymers with poly(ethylene glycol) and poly(propylene glycol) parts in the diol moiety and isocyanate functional groups at both terminals of the prepolymer - PVA-SbQ polyvinyl alcohol stilbazolium polymer     

Somatic Donor Cell Type Correlates with Embryonic,but Not Extra-Embryonic,Gene Expression in Postimplantation Cloned Embryos

The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (P<1.0×10^–26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.     

Activation and translocation of PKCdelta is necessary for VEGF-induced ERK activation through KDR in HEK293T cells

VEGF-KDR/Flk-1 signal utilizes the phospholipase C-gamma-protein kinase C (PKC)-Raf-MEK-ERK pathway as the major signaling pathway to induce gene expression and cPLA2 phosphorylation. However, the spatio-temporal activation of a specific PKC isoform induced by VEGF-KDR signal has not been clarified. We used HEK293T (human embryonic kidney) cells expressing transiently KDR to examine the activation mechanism of PKC. PKC specific inhibitors and human PKCdelta knock-down using siRNA method showed that PKCdelta played an important role in VEGF-KDR-induced ERK activation. Myristoylated alanine-rich C-kinase substrate (MARCKS) translocates from the plasma membrane to the cytoplasm depending upon phosphorylation by PKC. Translocation of MARCKS-GFP induced by VEGF-KDR stimulus was blocked by rottlerin, a PKCdelta specific inhibitor, or human PKCdelta siRNA. VEGF-KDR stimulation did not induce ERK phosphorylation in human PKCdelta-knockdown HEK293T cells, but co-expression of rat PKCdelta-GFP recovered the ERK phosphorylation. Y311/332F mutant of rat PKCdelta-GFP which cannot be activated by tyrosine-phosphorylation but activated by DAG recovered the ERK phosphorylation, while C1B-deletion mutant of rat PKCdelta-GFP, which can be activated by tyrosine-phosphorylation but not by DAG, failed to recover the ERK phosphorylation in human PKCdelta-knockdown HEK293T cell. These results indicate that PKCdelta is involved in VEGF-KDR-induced ERK activation via C1B domain.     

Taurine inhibits K+-Cl- cotransporter KCC2 to regulate embryonic Cl- homeostasis via with-no-lysine (WNK) protein kinase signaling pathway

GABA inhibits mature neurons and conversely excites immature neurons due to lower K(+)-Cl(-) cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl(-) homeostasis, which is required for normal brain development.     

Involvement of placental peptidases associated with renin-angiotensin systems in preeclampsia

Preeclampsia is characterized by pregnancy-induced hypertension accompanied with protein urea and generalized edema. Preeclampsia develops during the second half of pregnancy and resolves postpartum promptly, implicating the placenta as a primary cause in the disorder. Normal pregnancy is associated with reductions in arterial pressure and attenuated pressor response to exogenous infused angiotensin II (ANG II). In contrast, women with preeclampsia show the similar sensitivity to the pressor effect of ANG II as do non-pregnant women. To elucidate the involvement of placental peptidases associated with renin-angiotensin systems, we determined the localization of angiotensin-converting enzyme (ACE) and aminopeptidase A (AP-A), ANG II degrading enzyme, in the placenta and compared the expression of mRNA and protein in uncomplicated and preeclamptic placenta. In addition, AP-A expression in trophoblastic cells treated with ANG II and ACE expression in HUVECs under hypoxic condition were analyzed, respectively. The expression of both peptidases in the preeclamptic placenta was significantly higher than those from uncomplicated. ACE was primarily localized to venous endothelial cells of stem villous whereas AP-A expression was recognized in the trophoblast and pericytes of fetal arterioles and venules within stem villous. Hypoxia induced ACE expression in HUVECs while both hypoxia and ANG II evoked AP-A expression in trophoblast. These results suggested that hypoxic condition in preeclampsia induces ACE activation in feto-placental unit to maintain the fetal hemodynamics and placental AP-A plays a role as a component of the barrier of ANG II between mother and fetus.     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.     

Quantitative Mass Density Image Reconstructed from the Complex X-Ray Refractive Index

We demonstrate a new analytical X-ray computed tomography technique for visualizing and quantifying the mass density of materials comprised of low atomic number elements with unknown atomic ratios. The mass density was obtained from the experimentally observed ratio of the imaginary and real parts of the complex X-ray refractive index. An empirical linear relationship between the X-ray mass attenuation coefficient of the materials and X-ray energy was found for X-ray energies between 8 keV and 30 keV. The mass density image of two polymer fibers was quantified using the proposed technique using a scanning-type X-ray microbeam computed tomography system equipped with a wedge absorber. The reconstructed mass density agrees well with the calculated one.