Biosynthesis of rat insulin-like growth factor II. I. Immunochemical demonstration of a approximately 20-kilodalton biosynthetic precursor of rat insulin-like growth factor II in metabolically labeled BRL-3A rat liver cells

BRL-3A rat liver cells synthesize mature 7484-dalton rat insulin-like growth factor II (rIGF-II) as a approximately 22-kDa precursor, presumably prepro-rIGF-II. In the present study, we have biosynthetically labeled intact BRL-3A cells with [35S]cysteine and immunoprecipitated cell lysates and media with antisera to rIGF-II. A approximately 20-kDa protein was identified in immunoprecipitates of cell lysates having properties consistent with pro-rIGF-II. The approximately 20-kDa protein is precipitated by immune sera but not by nonimmune serum. Its immunoprecipitation is specifically inhibited by unlabeled rIGF-II but not by insulin. It is not precipitated from labeled lysates of a subclone of BRL-3A cells (BRL-3A2) that does not synthesize rIGF-II. The approximately 20-kDa protein is rapidly labeled intracellularly (10 min) but is not detected in BRL-3A media. In pulse-chase experiments, radioactivity in the approximately 20-kDa protein disappears during the chase and appears, at later times, in specifically immunoprecipitated approximately 19-, approximately 10-, approximately 8-, and approximately 7-kDa proteins in media and, to a limited extent, intracellularly. A protein with electrophoretic mobility identical to that of the approximately 20-kDa protein observed in cell lysates is immunoprecipitated from 35S-proteins whose synthesis is directed by BRL-3A RNA in a reticulocyte lysate cell-free translation system supplemented with microsomal membranes, and presumably arises by cotranslational removal of the signal peptide from approximately 22-kDa prepro-rIGF-II. Processing of the approximately 20-kDa protein in intact BRL-3A cells to intermediate and mature rIGF-II species appears to occur at the time of secretion and/or shortly thereafter, with the different forms appearing at approximately the same time.     

Relationship between ionic surroundings and insulin actions on glucose transport and Na,K-pump in muscles

1. It is well known that insulin has various effects on glucose transport and the Na,K-pump in muscles. It is also known to have some effects on the membrane potential--in general, insulin induces a hyperpolarization of the membrane in muscles. Furthermore, it is suggested that the actions of insulin are modified by changes in ionic surroundings. 2. In this review article, the actions of ionic surroundings and insulin on glucose transport in muscles are discussed; in particular, the effects of changes in extracellular and/or intracellular concentrations of Na, K, Ca and H ions will be mentioned. 3. The actions of ionic surroundings and insulin on the Na,K-pump in muscles are discussed; in particular, the effects of changes in extracellular an/or intracellular concentrations of Na, K, Ca and H ions will be examined. 4. The relationship between the actions of ionic surroundings and insulin are discussed. 5. In particular, the effects of changes in ionic surroundings on the insulin-induced hyperpolarization of the membrane are discussed by relating it to the Na,K-pump function. The relationship between the insulin-induced change in membrane potential and glucose transport will be also mentioned.     

Role of virus inhibitory factor or interferon in the antitumoral effect of whole-body hyperthermia

The interferon inducing effect of hyperthermia was studied in normal and tumor-bearing mice. Circulating interferon was temporarily detected one day after subcutaneous transplantation of 1.6 X 10(6) Ehrlich's ascites tumor cells. Hyperthermia of 43.5 degrees C for 5 min did not induce the interferon formation in mice with or without subcutaneous tumor of the cells. These findings showed that the induction of interferon formation was not main cause of the hyperthermia-induced tumor inhibition.     

Reductive activation of the coenzyme A/acetyl-CoA isotopic exchange reaction catalyzed by carbon monoxide dehydrogenase from Clostridium thermoaceticum and its inhibition by nitrous oxide and carbon monoxide

The final steps in the synthesis of acetyl-CoA by CO dehydrogenase (CODH) have been studied by following the exchange reaction between CoA and the CoA moiety of acetyl-CoA. This reaction had been studied earlier (Pezacka, E., and Wood, H. G. (1986) J. Biol. Chem. 261, 1609-1615 and Ramer, W. E., Raybuck, S. A., Orme-Johnson, W. H., and Walsh, C. T. (1989) Biochemistry 28, 4675-4680). The CoA/acetyl-CoA exchange activity was determined at various controlled redox potentials and was found to be activated by a one-electron reduction with half-maximum activity occurring at -486 mV. There is approximately 2000-fold stimulation of the exchange by performing the reaction at -575 mV relative to the rate at -80 mV. Binding of CoA to CODH is not sensitive to the redox potential; therefore, the reductive activation affects some step other than association/dissociation of CoA. We propose that a metal center on CODH with a midpoint reduction potential of less than or equal to -486 mV is activated by a one-electron reduction to cleave the carbonyl-sulfur bond and/or bind the acetyl group of acetyl-CoA. Based on a comparison of the redox dependence of this reaction with that for methylation of CODH (Lu, W-P., Harder, S. R., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3124-3133) and CO2 reduction and formation of the Ni-Fe-C EPR signal (Lindahl, P. A., Münck, E., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3873-3879), we propose that the assembly of the acetyl group of acetyl-CoA, i.e. binding the methyl group of the methylated corrinoid/iron-sulfur protein, binding CO, and methyl migration to form the acetyl-CODH intermediate, occur at the novel Ni-Fe3-4-containing site in CODH. CO has two effects on the CoA/acetyl-CoA exchange: it activates the reaction due to its reductive capacity and its acts as a noncompetitive inhibitor. We also discovered that the CoA/acetyl-CoA exchange was inhibited by nitrous oxide via an oxidative mechanism. In the presence of a low-potential electron donor, CODH becomes a nitrous oxide reductase which catalytically converts N2O to N2. This study combined with earlier results (Lu, W-P., Harder, S. R., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3124-3133) establishes that the two-subunit form of CODH is completely active in all reactions known to be catalyzed by CODH.     

Characterization of β-amylase and its deficiency in various rice cultivars

 β-Amylase deficiency in various cultivars of rice was examined at the molecular level. Using an antibody against β-amylase purified from germinating seeds of rice, we were able to demonstrate the expression and organization of the β-amylase gene in normal and deficient cultivars. Although β-amylase is a starch-hydrolyzing enzyme, as is α-amylase, the β-amylase protein/gene is expressed differently from the α-amylase protein/gene; i.e. (1) β-amylase is synthesized only in aleurone cells, (2) the enzyme production in the embryo-less half-seeds is not under hormonal control. We identified some cultivars of rice that are deficient for β-amylase activity. We present new evidence that synthesis is blocked at the level of mRNA synthesis in the deficient cultivars. The usefulness of β-amylase as a crop trait is also discussed. Received: 8 May 1998 / Accepted: 5 June 1998     

Pollen size-number trade-off and pollen-pistil relationships in Pedicularis (Orobanchaceae)

Current patterns of floral design in Pedicularis must have undergone an evolutionary process of interacting among components of floral traits, and then formed internal relationships among these traits. To detect such correlations, which may provide insight to understand flower evolution, 40 Pedicularis species representing all corolla types of the genus were studied. Results show that, interspecifically, pollen size correlates negatively with pollen number, but positively with pistil length. This suggests that plants evolve an optimal pollen size, which balances the advantages of large pollen size for gametophytic competition against the fecundity disadvantages of fewer pollen grains. In contrast to sex allocation theory, this study does not find a trade-off, but an interspecific positive correlation between pollen and ovule number. This is consistent with the hypothesis that genetic variation for resource acquisition may in part be responsible for the lack of negative correlation between male and female function.This work was supported by the State Key Basic Research and Development Plan, China (Grant No. G2000046804) to YHG. The authors would like to thank Peter K. Endress and two anonymous reviewers for providing critical comments and helpful suggestions, Qing-Feng Wang, Jing-Yuan Wang and Jin-Ming Chen for their helpful suggestions. Shi-Guo Sun, Jing Xia, and Qian Yu are thanked for their assistance in both the field work and laboratory phases of the project.     

Studies on Crocker sarcoma 180 under the effect of alkylating agents. IV. Morphological studies of sarcoma 180 during treatment with the antitumour drug cytoxan

The effect of one massive dose of cytoxan (150 mg/kg), as an alkylating agent, on the morphology of sarcoma 180 cells was followed after 1, 3, 6, 12, 24, 48, 96, 120, and 168 h. After 1 h the cells of treated tumour showed clumping and a decrease in their size. In addition, statistical analysis manifested significant decrease in all phases of mitotic figures. All mitotic figures were completely stopped after 3 h from cytoxan treatment. After 6 and 12 h from cytoxan treatment the hyaline degenerations became more marked in the treated tumour tissue. Chromosomal bridge formation, nuclei aberrations and polynuclear cells were more prominent in 24, 48, and 96 h from cytoxan treatment. Increase in the nuclear size and hyaline degeneration were observed in treated section of 120 and 168 h.     

Cryopreservation of spermatozoa of a transgenic mouse]

Spermatozoa of a homozygous transgenic mouse, in which the firefly luciferase gene was expressed under the control of beta-actin promoter, were frozen at -196 degrees C. One fourth of the frozen sperm was later thawed and used for in vitro fertilization. Thirty-six of 65 oocytes (55.4%) developed to the 2-cell stage. All the 2-cell embryos were transferred to the oviducts of pseudopregnant recipients and 23 young (63.9%, 23/36) were born. All of young analyzed carried the transgene and showed the luciferase gene expression.