Germanium oxide inhibits the transition from G2 to M phase of CHO cells

We report here for the first time that germanium oxide (GeO(2)) blocks cell progression. GeO(2) is not genotoxic to Chinese hamster ovary (CHO) cells and has limited cytotoxicity. However, GeO(2) arrests cells at G2/M phase. The proportion of cells stopped at G2/M phase increased dose-dependently up to 5 mM GeO(2) when treated for 12 h, but decreased at GeO(2) concentration was greater than 5 mM. Analysis of 5-bromodeoxyuridine-labeled cells indicated that GeO(2) delayed S phase progression in a dose-dependent manner, and blocked cells at G2/M phase. Microscopic examination confirmed that GeO(2) treatment arrested cells at G2 phase. Similar to several other events that cause G2 block, the GeO(2)-induced G2 block can also be ameliorated by caffeine in a dose- and time-dependent manner. To explore the mechanism of G2 arrest by GeO(2), cyclin content and cyclin-dependent kinase activity were examined. Cyclin B1 level was not affected after GeO(2) treatment in CHO cells. However, GeO(2) decreased p34(cdc2) kinase (Cdk1) activity. The kinase activity recovered within 9 h after GeO(2) removal and correlated with the transition of G2/M-G1 phase of the cells. This result suggests that GeO(2) treatment reduces Cdk1 activity and causing the G2 arrest in CHO cells.     

Regulation of MTP expression in developing swine

To define the developmental expression of microsomal triglyceride transfer protein (MTP) large subunit mRNA and protein, samples of small intestine and liver were collected from 40-day gestation fetal, 2-day-old newborn, 3-week-old suckling, and 2-month-old weanling swine. In fetal animals, MTP mRNA expression was high in intestine and liver. Postnatally, jejunal expression paralleled the intake of a high-fat breast milk diet and declined after weaning. Ileal expression was comparable with that of jejunum in 2-day-old animals, but declined to low levels afterward. Hepatic expression declined postnatally and remained low. MTP protein expression generally paralleled mRNA expression, except in fetal intestine in which no 97 kDa protein was detected. In 2-day-old piglets, a high-triacylglycerol diet increased jejunal and ileal MTP mRNA levels, as compared to a low-triacylglycerol diet. To test the roles of glucocorticoids and fatty acids in MTP regulation, a newborn swine enterocyte cell line (IPEC-1) was used. Except at day 2 of differentiation, dexamethasone did not influence MTP expression. Fatty acids either up-regulated or down-regulated MTP expression, depending on the specific fatty acid and duration of exposure. Although programmed genetic cues regulate MTP expression during development, clearly the amount and fatty acid composition of dietary lipid also play regulatory roles.     

Paclitaxel-resistant human ovarian cancer cells undergo c-Jun NH2-terminal kinase-mediated apoptosis in response to noscapine

We have previously discovered the opium alkaloid noscapine as a microtubule interacting agent that binds to tubulin, alters the dynamics of microtubule assembly, and arrests mammalian cells at mitosis (Ye, K., Ke, Y., Keshava, N., Shanks, J., Kapp, J. A., Tekmal, R. R., Petros, J., and Joshi, H. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1601-1606; Ye, K., Zhou, J., Landen, J. W., Bradbury, E. M., and Joshi, H. C. (2001) J. Biol. Chem. 276, 46697-46700; Zhou, J., Panda, D., Landen, J. W., Wilson, L., and Joshi, H. C. (2002) J. Biol. Chem. 277, 17200-17208). Here we show that noscapine does not compete with paclitaxel for tubulin binding and can efficiently inhibit the proliferation of both paclitaxel-sensitive and paclitaxel-resistant human ovarian carcinoma cells (i.e. the parental cell line 1A9 and two derivative cell lines, 1A9PTX10 and 1A9PTX22, which harbor beta-tubulin mutations that impair paclitaxel-tubulin interaction (Giannakakou, P., Sackett, D. L., Kang, Y. K., Zhan, Z., Buters, J. T., Fojo, T., and Poruchynsky, M. S. (1997) J. Biol. Chem. 272, 17118-17125). Strikingly, these cells undergo apoptotic death upon noscapine treatment, accompanied by activation of the c-Jun NH(2)-terminal kinases (JNK). Furthermore, inhibition of JNK activity by treatment with antisense oligonucleotide or transfection with dominant-negative JNK blocks noscapine-induced apoptosis. These findings thus indicate a great potential for noscapine in the treatment of paclitaxel-resistant human cancers. In addition, our results suggest that the JNK pathway plays an essential role in microtubule inhibitor-induced apoptosis.     

蛋白激酶A和蛋白激酶C对豚鼠心肌细胞延迟整流钾电流的作用

目的 :研究蛋白激酶A和蛋白激酶C对豚鼠心室肌细胞延迟整流钾电流 (Ik)的影响。方法 :采用电极内液浓度差扩散法进行细胞内给药 ,利用全细胞膜片箝技术测定单细胞Ik。结果 :cAMP15 0 μmol/L使Ik及Ik ,tail(pA/pF)从 13.7± 2 .1和 6 .1± 0 .3增至 18.5± 3.3和 6 .4± 2 .1(P <0 .0 1,n =6 ) ;8 CPT cAMP15 0 μmol/L使电流 (pA/pF)从 11.4± 1.8及 5 .3± 0 .6增至 17.9± 4 .0和 6 .2± 1.3,PKA的选择性抑制剂 6 2 2 1.0 μmol/L的可逆转二者的作用。cAMP使Ik的激活曲线左移 ,半激活电压 (V1/ 2 )从 2 3.3mV移至 18.7mV ,激活曲线斜率 (k)在用药前后变化较小。 10 μmol/LPMA可以分别使Ik和Ik ,tial(pA/pF)从 12 .9± 1.8和 5 .0± 1.7升至 2 3.7± 2 .8和 7.5±1.1。PMA使I V曲线幅值增加 ,并随去极化电压的升高其作用加强 ,同时PMA使通道的激活曲线k从 15 .3mV升到 2 5 .6mV ,但对V1/ 2 基本无影响。结论 :蛋白激酶A和蛋白激酶C均可增加豚鼠心肌细胞Ik,但二者作用特点有所不同     

Comparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state (13)C,(15)N, and (2)H NMR

The solid-state (13)C CP-MAS NMR spectra of biosynthetically labeled [(13)C(alpha)]Tyr, [(13)C(beta)]Tyr, and [(13)C(alpha)]Val silk fibroin samples of Bombyx mori, in silk I (the solid-state structure before spinning) and silk II (the solid-state structure after spinning) forms, have been examined to gain insight into the conformational preferences of the semicrystalline regions. To establish the relationship between the primary structure of B. mori silk fibroin and the "local" structure, the conformation-dependent (13)C chemical shift contour plots for Tyr C(alpha), Tyr C(beta), and Val C(alpha) carbons were generated from the atomic coordinates of high-resolution crystal structures of 40 proteins and their characteristic (13)C isotropic NMR chemical shifts. From comparison of the observed Tyr C(alpha) and Tyr C(beta) chemical shifts with those predicted by the contour plots, there is strong evidence in favor of an antiparallel beta-sheet structure of the Tyr residues in the silk fibroin fibers. On the other hand, Tyr residues take a random coil conformation in the fibroin film with a silk I form. The Val residues are likely to assume a structure similar to those of Tyr residues in silk fiber and film. Solid-state (2)H NMR measurements of [3,3-(2)H(2)]Tyr-labeled B. mori silk fibroin indicate that the local mobility of the backbone and the C(alpha)-C(beta) bond is essentially "static" in both silk I and silk II forms. The orientation-dependent (i.e., parallel and perpendicular to the magnetic field) solid-state (15)N NMR spectra of biosynthetically labeled [(15)N]Tyr and [(15)N]Val silk fibers reveal the presence of highly oriented semicrystalline regions.     

Structural analysis of an insect lysozyme exhibiting catalytic efficiency at low temperatures

Bombyx mori lysozyme (BmLZ), from the silkworm, is an insect lysozyme. BmLZ has considerable activity at low temperatures and low activation energies compared with those of hen egg white lysozyme (HEWLZ), according to measurements of the temperature dependencies of relative activity (lytic and glycol chitin) and the estimation of activation energies using the Arrhenius equation. Being so active at low temperatures and low activation energies is characteristic of psychrophilic (cold-adapted) enzymes. The three-dimensional structure of BmLZ has been determined by X-ray crystallography at 2.5 A resolution. The core structure of BmLZ is similar to that of c-type lysozymes. However, BmLZ shows some distinct differences in the two exposed loops and the C-terminal region. A detailed comparison of BmLZ and HEWLZ suggests structural rationalizations for the differences in the catalytic efficiency, stability, and mode of activity between these two lysozymes.     

Regulation of yeast actin behavior by interaction of charged residues across the interdomain cleft

His(73) participates in the regulation of the nucleotide binding cleft conformation in yeast actin. Earlier molecular dynamics studies suggested that Asp(184) interacts with His(73) thereby stabilizing a "closed-cleft" G-actin. However, beta-actin in the open-cleft state shows a closer interaction of His(73) with Asp(179) than with Asp(184). We have thus assessed the relative importance of Asp(184) and Asp(179) on yeast actin stability and function. Neutral substitutions at 184 or 179 alone had little adverse effect on the monomer and polymerization behavior of actin. Arg or His at 184 in H73E actin partially rescued the monomeric properties of H73E actin, as demonstrated by near-normal thermostability and wild-type (WT)-like protease digestion patterns. ATP exchange was still considerably faster than with WT-actin although slower than that of H73E alone. However, polymerization of H73E/D184R and H73E/D184H is worse than with H73E alone. Conversely, D179R rescued all monomeric properties of H73E to near WT values and largely restored polymerization rate and filament thermostability. These results and new simulations of G-actin in the "open" state underscore the importance of the His(73)-Asp(179) interaction and suggest that the open and not the closed state of yeast actin may be favored in the absence of the methyl group of His(73).     

Membrane polyunsaturated fatty acids and CSF cytokines in patients with schizophrenia

Findings to date provide evidence that altered membrane structure and function are present in patients with either first-episode or chronic schizophrenia, suggesting defects in phospholipid metabolism and cell signaling in schizophrenia. The purpose of this investigation is to test whether decreased membrane polyunsaturated fatty acids (PUFAs) were associated with an increased secretion of proinflammatory cytokines. Thus, we measured interleukin 6 (IL-6) and interleukin 10 (IL-10) in cerebrospinal fluid (CSF) of patients with chronic schizophrenia as well as PUFAs of red blood cell (RBC) membranes from the same individuals. A significant and inverse correlation was found between CSF IL-6 (not IL-10) and RBC membrane PUFAs levels in both haloperidol-treated and medication-free patients with schizophrenia. Specifically, such an association was found in the n-6 (18:2, 20:4, and 22:4) and, to a lesser extent, the n-3 fatty acids. Taken together, the present findings suggest that decreased membrane PUFAs may be related to an immune disturbance in schizophrenia, possibly resulting from an increased phospholipase A2 activity mediated through the proinflammatory cytokines.