Abstracts of papers (2001)
Last Update: 09/07/2001
Abstracts of papers (2001)
[2001-1] Tomitori,
H. et al., Biolchem. J. 353, 681-688 (2001)
We recently identified a gene (TPO1,
YLL028w) that encodes a polyamine transport protein on
the vacuolar membrane in yeast [Tomitori, Kashiwagi, Sakata,
Kakinuma and Igarashi (1999) J. Biol. Chem. 274, 3265-3267].
Because the existence of one or more other genes for a polyamine
transport protein on the vacuolar membrane was expected, we searched
sequence databases for homologues of the protein encoded by TPO1.
Membrane proteins encoded by the open reading frames YGR138c
(TPO2), YPR156c (TPO3) and YOR273c
(TPO4) were postulated to be polyamine transporters and,
indeed, were subsequently shown to be polyamine transport proteins
on the vacuolar membrane. Cells overexpressing these genes were
resistant to polyamine toxicity and showed an increase in polyamine
uptake activity and polyamine content in vacuoles. Furthermore,
cells in which these genes were disrupted showed an increased
sensitivity to polyamine toxicity and a decrease in polyamine
uptake activity and polyamine content in vacuoles. Resistance
to polyamine toxicity in cells overexpressing the genes was overcome
by bafilomycin A1, an inhibitor of the vacuolar H+-ATPase. Among
the four polyamine transporters, those encoded by TPO2
and TPO3 were specific for spermine, whereas those encoded
by TPO1 and TPO4 recognized spermidine and spermine.
These results suggest that polyamine content in the cytoplasm
of yeast is elaborately regulated by several polyamine transport
systems in vacuoles. Furthermore, it was shown that Glu-207,
Glu-324 (or Glu-323) and Glu-574 of TPO1 protein were important
for the transport activity.
[2001-2] Kawano,
M. et al., Arch. Microbiol. 175, 41-45 (2001)
Two high-affinity K+ uptake systems, KtrI
and KtrII, have been reported in Enterococcus hirae. A
mutant, JEMK1, defective in these two systems did not grow at
pH 10 in low-K+ medium (less than 1 mM K+), but grew well when
supplemented with 10 mM KCl. In this mutant, we found an energy-dependent
K+ uptake at pH 10 with a low affinity for K+ (Km of ~20
mM) and an extremely high rate [Vmax of 1.6 オmol min-1
(mg protein)-1]. Rb+ uptake [Km of ~40 mM and Vmax of 0.5 オmol min-1 (mg protein)-1], which was inhibited competitively by K+ and less prominently by Cs+, was also observed. The specificity of this transport is likely to be K+>Rb+>Cs+. This peculiar K+ transport plays a role as a salvage mechanism against defects in high-affinity systems in the K+ homeostasis of this bacterium.
[2001-3] Nitta,
T. et al., Exp. Cell Res. 265, 174-183 (2001)
The B cell lymphoma WEHI231 has been used
as a model for studying clonal deletion of B cells on the basis
of its ability to undergo growth arrest and apoptosis by B cell
antigen receptor (BCR) cross-linking. To comprehensively analyze
the genes involved in BCR-mediated apoptosis, we applied the
technique of serial analysis of gene expression (SAGE) to WEHI231.
Comparison of expression patterns revealed that BCR cross-linking
caused coordinate changes in the expression of genes involved
in polyamine metabolism. Polyamines are ubiquitous compounds
required for cell proliferation and homeostasis. The coordinate
expression of the polyamine-related genes was confirmed by semiquantitative
reverse transcriptase-polymerase chain reaction analysis. During
apoptosis, the genes involved in polyamine biosynthesis were
downregulated, whereas those involved in polyamine catabolism
were upregulated, suggesting that intracellular polyamines play
a role in BCR-mediated apoptosis. Levels of intracellular putrescine,
spermidine, and spermine were reduced after BCR cross-linking.
These effects were prevented by concurrent CD40 stimulation,
which blocked BCR-mediated apoptosis. Furthermore, addition of
spermine could repress the BCR-mediated apoptosis by attenuating
the mitochondrial membrane potential (Dym) loss and activation of caspase-7 induced by BCR signaling. These findings
strongly suggest that polyamine regulation is involved in apoptosis during
B cell clonal deletion.
[2001-4] Sharmin,
S. et al., Biochem. Biophys. Res. Commun. 282,
228-235 (2001)
The toxicity of extracellular spermine,
determined in the presence of fetal calf serum, was studied using
three cell lines: FM3A, L1210, and NIH3T3 cells. Amine oxidase
in fetal calf serum produces aminodialdehyde generating acrolein
spontaneously, H2O2, and ammonia from spermine. Spermine toxicity
was prevented by aldehyde dehydrogenase, but not by catalase.
Similar concentrations of spermine and acrolein were needed to
produce toxicity. Other aldehydes (formaldehyde, acetaldehyde,
and propionaldehyde) and hydrogen peroxide were less toxic than
acrolein. Spermidine and 3-aminopropanal, which produces acrolein,
also exhibited severe cytotoxicity. The degree of cytotoxicity
of spermine, spermidine, and 3-aminopropanal was nearly parallel
with the amount of acrolein produced from each compound. Thus,
it was deduced that acrolein is a major toxic compound produced
from polyamines (spermine and spermidine) by amine oxidase.
[2001-5] Murata,
T. et al., Biochim. BIOphys. Acta 1505, 75-81
(2001)
V-ATPases make up a family of proton pumps
distributed widely from bacteria to higher organisms. We found
a variant of this family, a Na(+)-translocating ATPase, in a
Gram-positive bacterium, Enterococcus hirae. The Na(+)-ATPase
was encoded by nine ntp genes from F to D in an ntp
operon (ntpFIKECGABDHJ): the ntpJ gene encoded
a K(+) transporter independent of the Na(+)-ATPase. Expression
of this operon, encoding two transport systems for Na(+) and
K(+) ions, was regulated at the transcriptional level by intracellular
Na(+) as the signal. Structural aspects and catalytic properties
of purified Na(+)-ATPase closely resembled those of other V-type
H(+)-ATPases. Interestingly, the E. hirae enzyme showed
a very high affinity for Na(+) at catalytic reaction. This property
enabled the measurement of ion binding to this ATPase for the
first time in the study of V- and F-ATPases. Properties of Na(+)
binding to V-ATPase were consistent with the model that V-ATPase
proteolipids form a rotor ring consisting of hexamers, each having
one cation binding site. We propose here a structure model of
Na(+) binding sites of the enzyme.
[2001-6] Yoshida,
M. et al., J. Biol. Chem. 276, 16289-16295
(2001)
The effects of polyamines on the synthesis
of various sigma subunits of RNA polymerase were studied using Western blot analysis.
Synthesis of sigma28
was stimulated 4.0-fold and that of sigma38 was stimulated 2.3-fold by polyamines, whereas
synthesis of other sigma subunits was not influenced by polyamines. Stimulation of sigma28 synthesis was due
to an increase in the level of cAMP, which occurred through polyamine
stimulation of the synthesis of adenylate cyclase at the level
of translation. Polyamines were found to increase the translation
of adenylate cyclase mRNA by facilitating the UUG codon-dependent
initiation. Analysis of RNA secondary structure suggests that
exposure of the Shine-Dalgarno sequence of mRNA is a prerequisite
for polyamine stimulation of the UUG codon-dependent initiation.
[2001-7] Igarashi,
K. et al., Res. Microbiol. 152, 271-278 (2001)
The polyamine content of cells is regulated
by biosynthesis, degradation, and transport. In Escherichia
coli, the genes for three different polyamine transport systems
have been cloned and characterized. Two uptake systems (putrescine-specific
and spermidine-preferential) are ABC transporters, each consisting
of a periplasmic substrate binding protein, two transmembrane
proteins, and a membrane-associated ATPase. The third transport
system, catalyzed by PotE, mediates both uptake and excretion
of putrescine. In this article, the properties of the first two
polyamine uptake systems are reviewed in detail.
[2001-8] Raj, V.
S. et al., J. Bacteriol. 183, 4493-4498 (2001)
Escherichia coli
CAG2242 cells are deficient in the speG gene encoding
spermidine acetyltransferase. When these cells were cultured
in the presence of 0.5 to 4 mM spermidine, their viability was
greatly decreased through the inhibition of protein synthesis
by overaccumulation of spermidine. When the cells were cultured
with a high concentration of spermidine (4 mM), a revertant strain
was obtained. We found that a 55-kDa protein, glycerol kinase,
was overexpressed in the revertant and that synthesis of a ribosome
modulation factor and the RNA polymerase sigma38 subunit, factors important for cell viability,
was increased in the revertant. Levels of L-glycerol 3-phosphate
also increased in the revertant. Transformation of glpFK,
which encodes a glycerol diffusion facilitator (glpF)
and glycerol kinase (glpK), to E. coli CAG2242
partially prevented the cell death caused by accumulation of
spermidine. It was also found that L-glycerol 3-phosphate inhibited
spermidine binding to ribosomes and attenuated the inhibition
of protein synthesis caused by high concentrations of spermidine.
These results indicate that L-glycerol 3-phosphate reduced the
binding of excess amounts of spermidine to ribosomes so that
protein synthesis is recovered.
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