Abstracts of papers (2000)

Last Update: 11/14/2000

Abstracts of papers (2000)

[2000-1] Sakata, K. et al., Biolchem. J. 347, 297-303 (2000)

The regulation of polyamine transport by antizyme, a protein that is involved in the rapid degradation of ornithine decarboxylase (ODC), was studied in FM3A mouse cells overproducing ODC. Both artificial (Z1) and natural antizymes not only inhibited polyamine uptake but also stimulated polyamine excretion. The properties of the polyamine transporter regulated by antizyme were characterized. The uptake of radiolabelled polyamines was inhibited by excess acetylpolyamines and a protonophore, CCCP (carbonyl cyanide m-chlorophenylhydrazone), whereas the excretion of radiolabelled polyamines was stimulated by unlabelled polyamines, acetylpolyamines and CCCP in the medium. Furthermore, it is shown that polyamines and acetylpolyamines are excreted from cells. On the basis of the results, it is discussed how antizyme regulates polyamine transport negatively.

[2000-2] Moroki, Y. et al., Zool. Sci. 17, 191-200 (2000)

Low molecular signalling molecules such as cAMP and cGMP are expected to have important function in early morphogenetic processes in animal deveolopment. We examined the effect of 8-bromo-cyclic GMP (Br-cGMP) on Xenopus embryogenesis, using 8-bromo-cyclic AMP (Br-cAMP) as a reference. When Xenopus gastrulae were cultured in the medium which contained these analogues, their development was affected in specific and dosage-dependent manners: While Br-cAMP induced anomaly only in head part (swelling of myelencephalon with enlarged ventricle), Br-cGMP induced shortening in body length often accompanied by bending of the cephalo-caudal axis. In embryos treated with Br-cGMP at a high dose, cellular movement was inhibited as revealed by SEM and this resulted in the formation of tadpoles with unclosed yolk plug. Br-cGMP at lower doses induced severe inhibition of the development of notochord and muscles. Since HPLC analyses revealed that both analogues were uptaken into embryonic cells, we assumed that the morphological effects observed were induced by the interference of the normal functioning of cGMP and cAMP, respectively, by Br-cGMP and Br-cAMP. Based on the results obtained, we assume that while cGMP is involved mainly in the differentiation of mesodermal structures, especially in formation of notochord and muscles, cAMP is involved mainly in the defferentiation of neural structures.

[2000-3] Murata, T. et al., J. Biol. Chem. 275, 13415-13419 (2000)

Rotation catalysis theory has been successfully applied to the molecular mechanism of the ATP synthase (F0F1-ATPase) and probably of the vacuolar ATPase. We investigated the ion binding step to Enterococcus hirae Na+-translocating V-ATPase. The kinetics of Na+ binding to purified V-ATPase suggested 6 +/- 1 Na+ bound/enzyme molecule, with a single high affinity (Kd(Na+) = 15 +/- 5 [micro]M). The number of cation binding sites is consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. Release of the bound [22]Na+ from purified molecules in a chasing experiment showed two phases: a fast component (about two-thirds of the total amount of bound Na+; kexchange > 1.7 min-1) and a slow component (about one-third of the total; kexchange = 0.16 min-1), which changes to the fast component by adding ATP of ATP[gamma]S. This suggested that about two-thirds of the Na+ binding sites of the Na+-ATPase are readily accessible from the aqueous phase and that the slow component is important for the transport reaction.

[2000-4] Kawano, M. et al., J. Bacteriol. 182, 2507-2512 (2000)

The ntpJ gene, a cistron located at the tail end of the vacuolar-type Na+-ATPase (ntp) operon of Enterococcus hirae, encodes a transporter of the KtrII K+ uptake system. We found that K+ accumulation in the ntpJ-disrupted mutant JEM2 was markedly enhanced by addition of valinomycin at pH 10. Studies of the membrane potential (DY; inside negative) by 3,3'-dihexyloxacarbocyanine iodide fluorescence revealed that the DY was hyperpolarized at pH 10 in JEM2; the DY values of the parent strain ATCC9790 and JEM2, estimated by determining the equilibrium distribution of K+ or Rb+ in the presence of valinomycin, were -118 and -160 mV, respectively. DY generation at pH 10 was accomplished by an electrogenic Na+ efflux via the Na+-ATPase, whose levels in the two strains were quite similar. Na+ uptake driven by an artificially imposed DY (inside negative) was missing in JEM2, suggesting that NtpJ mediates Na+ movement in addition to K+ movement. Finally, the growth of JEM2 arrested in K+-limited high-Na+ medium at pH 10 was restored by addition of valinomycin. These results suggest that NtpJ mediates electrogenic transport of K+ as well as Na+, that it likely mediates K+ and Na+ cotransport, and that Na+ movement via NtpJ is the major Na+ reentry pathway at high pH values.

[2000-5] Shiokawa, K. et al., Comp. Biochem. Physiol. Part B 126, 149-155 (2000)

When we studied polyamine metabolism in Xenopus embryos, we cloned the cDNA for Xenopus S-adenosylmethionine decarboxylase (SAMDC), which converts SAM (S-adenosylmethionine), the methyl donor, into decarboxylated SAM (dcSAM), the aminopropyl donor, and microinjected its in vitro transcribed mRNA into Xenopus fertilized eggs. We found here that the mRNA injection induces a SAM deficient state in early embryos due to over-function of the overexpressed SAMDC, which in turn induces inhibition of protein synthesis. Such embryos developed quite normally until blastula stage, but stopped development at the early gastrula stage, due to induction of massive cell dissociation and cell autolysis, irrespective of the dosage and stage of the mRNA injection. We found that the dissociated cells were TUNEL-positive, contained fragmented nuclei with ladder-forming DNA, and furthermore, rescued completely by coinjection of Bcl-2 mRNA. Thus, overexpression of SAMDC in Xenopus embryos appeared to switch on apoptotic program, probably via inhibition of protein synthesis. Here, we briefly review our results together with those reported from other laboratories. After discussing the general importance of this newly discovered apoptotic program, we propose that the maternal program of apoptosis serves as a surveillance mechanism to eliminate metabolically severely-damaged cells and functions as a 'fail-safe' mechanism for normal development in Xenopus embryos.

[2000-6] Igarashi, K. et al., Biochem. Biophys. Res. Commun. 271, 559-564 (2000)

In recent years the functions of polyamines (putrescine, spermidine, and spermine) have been studied at the molecular level. Polyamines can modulate the functions of RNA, DNA, nucleotide triphosphates, proteins, and other acidic substances. A major part of the cellular functions of polyamines can be explained through a structural change of RNA which occurs at physiological concentrations of Mg2+ and K+ because most polyamines exist in a polyamine-RNA complex within cells. Plyamines were found to modulate protein synthesis at several different levels including stimulation of special kinds of protein synthesis, stimulation of the assembly of 30 S ribosomal subunits and stimulation of Ile-tRNA formation. Effects of polyamines on ion channels have also been reported and are gradually being clarified at the molecular level.

[2000-7] Kai, M. et al., Int. J. Develop. Biol. 44, 507-510 (2000)

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) mRNA in 1- and 2-cell stage Xenopus embryos induces cell autonomous dissociation at the late blastula stage and developmental arrest at the early gastrula stage. The induction of cell dissociation took place "punctually" at the late blastula stage in the SAMDC-overexpressed cells, irrespective of the stage of the microinjection of SAMDC mRNA. When we examined the cells undergoing the dissociation, we found that they are TUNEL-positive, contain fragmented nuclei with condensed chromatin and fragmented DNA. Furthermore, by injecting Xenopus Bcl-2 mRNA together with SAMDC mRNA, we showed that SAMDC-overexpressed embryos are rescued completely by Bcl-2 and become tadpoles. These results indicate that cell dissociation induced by SAMDC overexpression is due to apoptotic cell death. Since the level of S-adenosylmethionine (SAM) is greatly reduced in SAMDC-overexpressed embryos and this induces inhibition of protein synthesis accompanied by the inhibition of DNA and RNA syntheses, we conclude that deficiency in SAM induced by SAMDC overexpression activates the maternal program of apoptosis in Xenopus embryos at the late blastula stage, but not before. We propose that this mechanism serves as a surveillance mechanism to check and eliminate cells physiologically damaged during cleavage stage.

[2000-8] Hayashi, S. et al., Biochem. Biophys. Res. Commun. 277, 117-123 (2000)

The role of eIF4G during the initiation of protein synthesis was studied using mouse mammary carcinoma FM3A cells and FM4G cells that overproduce an N-terminarlly truncated form of eIF4G, which lacks the binding site of poly(A)-binding protein. An increase in eIF4G was correlated with an increase in protein synthesis and RNA helicase activity. Translation of mRNAs having both short and long 5'-untranslated regions (5'-UTR) increased significantly in FM4G cells compared to that in FM3A cells. Both full-length and N-terminally truncated eIF4G transfectants of NIH3T3 cells formed colonies in soft agar and increased the saturation density of cell growth, indicating that both eIF4Gs function similarly. We also found that an internal ribosome entry site (IRES) exists in the 5'-UTR of ornithine decarboxylase mRNA and that IRES-dependent protein synthesis increased in FM4G cells. Our results indicate that an increase in eIF4G contributes to the formation of active eIF4F similarly to that caused by an increase in eIF4E, as well as to a stimulation of IRES-dependent protein synthesis.

[2000-9] Ikegami, M. et al., Biosci. Biotechnol. Biochem. 64, 1088-1092 (2000)

Enterococcus hirae grows in a broad pH range from 5 to 11. An E. hirae mutant 7683 lacking the activities of two sodium pumps, Na+-ATPase and Na+/H+ antiporter, does not grow in high Na+ medium at pH above 7.5. We found that 7683 grew normally in high Na+ medium at pH 5.5. Although an energy-dependent sodium extrusion at pH 5.5 was missing, the intracellular levels of Na+ and K+ were normal in this mutant. The Na+ influx rates of 7683 and two other strains at pH 5.5 were much slower than those at pH 7.5. These results suggest that Na+ elimination of this bacterium at acid pH is achieved by a decrease in Na+ entry and a normal K+ uptake.

[2000-10] Kashiwagi, K. et al., J. Biol. Chem. 275, 36007-36012 (2000)

The PotE protein can catalyze both uptake and excretion of putrescine. The Km values of putrescine for uptake and excretion are 1.8 and 73 [micro]M, respectively. Uptake of putrescine is dependent on the membrane potential, whereas excretion involves putrescine-ornithine antiporter activity. Amino acids involved in both activities were identified using mutated PotE proteins. It was found that Cys62, Trp201, Trp292, and Tyr425 were strongly involved in both activities, and that Tyr92, Cys210, Cys285, and Cys286 were moderately involved in the activities. Mutations of Tyr78, Trp90, and Trp422 mainly affected uptake activity and the Km values for putrescine uptake by these PotE mutants increased greatly, indicating that these amino acids are involved in the high affinity uptake of putrescine by PotE. Mutations of Lys301 and Tyr308 mainly affected excretion activity (putrescine-ornithine antiporter activity), and excretion by these mutants was not stimulated by ornithine, indicating that these amino acids are involved in the recognition of ornithine. It was found that the putrescine and ornithine recognition site on PotE is located at the cytoplasmic surface and the vestibule of the pore consisting of 12 transmembrane segments. Based on the results of competition experiments with various putrescine analogues and the disulfide cross-linking of PotE between cytoplasmic loops and the COOH terminus, a model of the putrescine recognition site on PotE consisting of the identified amino acids is presented.