Tomohito Kakegawa

pH dependence of the function of sodium ion extrusion systems in Escherichia coli

Escherichia coli has three systems for sodium ion extrusion, NhaA, NhaB and ChaA. In this study, we examined the effect of pH on the function of these transporters using mutants having one of them, and found that (1) a mutant having NhaB excreted sodium ions at pH 7.5 but not at pH 8.5, (2) the efflux of sodium ions from mutant cells having ChaA was observed at both pH 7.5 and 8.5, but the activity was lower at pH 7.5, and (3) sodium ions were excreted from mutant cells having NhaA at pH 6.5 to 8.5. The extrusion activity of cells having NhaA was higher than that of cells having NhaB or ChaA. These results indicate that NhaB functions at a pH below 8, and ChaA extrudes sodium ions mainly at an alkaline pH above 8. It was also suggested that the activity of NhaB and ChaA is not enough to maintain a low level of internal sodium ions when the external concentration of sodium ions is high, and NhaA is induced within a wide range of medium pH under such conditions.

Polyamine binding sites on Escherichia coli ribosomes

To determine the binding sites of polyamines on Escherichia coli ribosomes, ribosomal proteins crosslinked with polyamines through bifunctional reagents were analyzed. When 1,5-difluoro-2,4-dinitrobenzene was used as a crosslinking reagent, spermine was bound to S3, S8, S9, L1, L2, L3, L5, L6, L13, L18, L24, and L27 proteins. When dimethyl suberimidate was used, spermine was bound to S1, S3, S4, S5, S7, S8, S9, S15, L1, L2, L3, L6, L18, and L24 proteins. In addition to crosslinking with the above proteins, spermidine, when crosslinked with dimethyl suberimidate, bound to S2, S14, S20, L4, L5, L9, L13, and L16 proteins. The relationship between the binding site(s) of polyamines on ribosomes and the function of polyamines is discussed.

Species difference in enantioselectivity for the oxidation of propranolol by cytochrome P450 2D enzymes.

We examined and compared enantioselectivity in the oxidation of propranolol (PL) by liver microsomes from humans and Japanese monkeys (Macaca fuscata). PL was oxidized at the naphthalene ring to 4-hydroxypropranolol, 5-hydroxypropranolol and side chain N-desisopropylpropranolol by human liver microsomes with enantioselectivity of [R(+)>S(-)] in PL oxidation rates at substrate concentrations of 10 microM and 1 mM. In contrast, reversed enantioselectivity [R(+)<S(-)] in PL 5-hydroxylation and N-desalkylation rates at the same substrate concentrations was observed in monkey liver microsomes, although the selectivity was the same for PL 4-hydroxylation between the two species. All oxidation reactions of the PL enantiomers in human liver microsomes showed biphasic kinetics, i.e. the reactions could be expressed as the summation of a low-K(m) phase and a high-K(m) phase. Inhibition studies using antibodies and characterization of CYP2D6 enzymes expressed in insect cells or human lymphoblastoid cells indicated that the enantioselectivity of PL oxidation, especially the ring 4- and 5-hydroxylations reflected the properties of CYP2D6 in human liver microsomes. In monkey liver microsomes, all of the oxidation reactions of S(-)-PL showed biphasic kinetics, whereas ring 4- and 5-hydroxylations were monophasic and side chain N-desisopropylation was biphasic for R(+)-PL. Similarly, from the results of inhibition studies using antibodies and inhibitors of cytochrome P450 (P450), it appears that the reversed selectivity [R(+)<S(-)] of PL oxidation rates is catalyzed by CYP2D enzyme(s) in monkey liver at low substrate concentrations. These results indicate that different properties of P450s belonging to the 2D subfamily cause the reversed enantioselectivity between human and monkey liver microsomes.

Expression of chaA, a sodium ion extrusion system of Escherichia coli, is regulated by osmolarity and pH

ChaA, one of the sodium ion extrusion systems of Escherichia coli, was found to function at high pH [Biochim. Biophys. Acta 1363 (1998) 231]. A chaA-lacZ transcriptional fusion gene was constructed using chaA of E. coli O157:H7 and its expression was observed in strains derived from E. coli K12. The fusion gene was expressed at high pH and was induced by the addition of NaCl, KCl or sucrose. The amount of chaA mRNA measured by reverse transcription-polymerase chain reaction (RT-PCR) was increased by the addition of sucrose to alkaline growth medium. These results suggested that chaA expression was regulated by medium osmolarity and pH.

Different proteins are phosphorylated under acidic environments in Jurkat cells

In order to test the ability of immune cells to grow under acidic conditions, Jurkat cells were cultured in medium at pH 6.3. At this acidic pH, the cells required approximately 2 days per generation, while the generation time was approximately 1 day for cells grown in near neutral medium. Western blot analysis using anti-phosphotyrosine antibody (4G10) showed that protein phosphorylation was affected by external pH. For example, the level of a 66-kDa phosphoprotein increased dramatically in acidic medium, whereas the amount of a 34-kDa phosphoprotein decreased. These results suggest that Jurkat cells use different signal pathways under acidic environments.

Effect of various polyamine analogs on in vitro polypeptide synthesis.

Various polyamine analogs were examined for their ability to stimulate and to function as sparing agents for the Mg2+ requirement in polypeptide synthesis at various temperatures in Escherichia coli (37 and 47 degrees C) and the extremely thermophilic Thermus thermophilus (60 and 70 degrees C) cell-free systems. The optimal concentration of each polyamine analog increased as the incubation temperature was elevated. At a fixed temperature, the optimal concentration of polyamine analogs was in the order diamines greater than triamines greater than tetraamines greater than pentaamines. All diamines tested stimulated polypeptide synthesis almost equally but lowered the optimal Mg2+ concentration in the order diaminopropane greater than putrescine greater than cadaverine. The degree of diamine stimulation was maximal at 37 degrees C. The effects of three triamines were very similar in the E. coli system but in the T. thermophilus system spermidine was most effective in stimulation of polypeptide synthesis. From the results of experiments using tetraamines and pentaamines, it was deduced that the presence of both aminobutyl and aminopropyl groups in polyamine analogs is important for stimulation of polypeptide synthesis. In the E. coli system, triamines were the most effective polyamines for stimulation of polyphenylalanine synthesis at both 37 and 47 degrees C, while, in the T. thermophilus system, thermospermine, a tetraamine, was most effective at 60 degrees C and 3,4,4,3-pentaamine was most effective at 70 degrees C.

An IκB-β COOH terminal region protein is essential for the proliferation of CHO cells under acidic stress

CHO‐K1 cells were able to proliferate and maintain pHi homeostasis at pH 6.3. A novel acidic sensitive mutant, AS‐5B, which proliferated at pH 7.4 but failed to either proliferate or maintain pHi homeostasis at pH 6.3, was derived from CHO‐K1 using a replica method. The acidic‐sensitivity of AS‐5B was not due to deficiencies in sodium proton exchangers, HCO  3− (co)transporters or H+‐ATPases. A cDNA clone encoding a COOH terminal region of IκB‐β conferred partial acidic‐resistance on AS‐5B, and the encoded protein was present in CHO‐K1, but was nearly absent from AS‐5B. Our data demonstrated that the expression of this small protein was essential for the proliferation of CHO cells under acidic stress.

Stabilization of 30 S ribosomal subunits of Bacillus subtilis W168 by spermidine and magnesium ions

An explanation for the fragility of 30 S ribosomal subunits of Bacillus subtilis has been studied. Degradation of 16 S ribosomal RNA, rather than degradation of ribosomal proteins, was found to cause the inactivation of 30 S subunits. Although RNAases were bound specifically to 30 S ribosomal subunits, the RNAases were able to function. Spermidine was found to contribute to the stabilization of 30 S ribosomal subunits by inhibiting the degradation of 16 S ribosomal RNA. A high concentration of Mg2+ also stabilized the 30 S ribosomal subunits of Bacillus subtilis. The polypeptide synthetic activity of 30 S ribosomal subunits prepared in the presence of spermidine was at least 4-times greater than that of 30 S ribosomal subunits prepared in the absence of spermidine; this activity was maintained without any loss for 3 months at -70 degrees C.

Effects of macrocyclic polyamines and polymethylenediamines on reactions influenced by polyamines.

The effects of macrocyclic polyamines and polymethylenediamines on various reactions influenced by polyamines have been studied. Among the amines tested, 2,3,4,3- and 3,3,3,4-cyclic polyamines, NH2(CH2)6NH2 and NH2(CH2)8NH2 had some ability to stimulate polyphenylalanine synthesis, globin synthesis and rat liver isoleucyl-tRNA formation. The degree of stimulation was at most 40% of that obtained by polyamines. In the degradation of poly(C) by bovine pancreatic RNAase A, all tested amines stimulated the degradation. In the NADPH-dependent lipid peroxidation of rat liver microsomes, the degree of inhibition by 2,3,2,3- or 2,3,3,3-cyclic polyamine was greater than that by spermine. The hydrolysis of ATP by an oligomycin-sensitive ATPase was inhibited by 2,3,4,3- and 3,3,3,4-cyclic polyamines, NH2(CH2)10HN2 and spermine at somewhat comparable levels. None of the macrocyclic polyamines or polymethylenediamines stimulated the growth of a polyamine-requiring mutant of Escherichia coli. Possible explanations for the differences in the effects of amines on the various reactions are discussed.

Induction of differentiation of human myeloid leukemia cells by novel synthetic neurotrophic pyrimidine derivatives

OBJECTIVE Some pyrimidine analogues have been found to induce differentiation of several human myeloid leukemia cells. Newly synthesized heterocyclic pyrimidine derivatives promote neurite outgrowth and survival in neuronal cell lines. In this study, the growth-inhibiting and differentiation-inducing effects of these pyrimidine derivatives on human myeloid leukemia cells were examined. MATERIALS AND METHODS Several myeloid leukemia cells were cultured with novel heterocyclic pyrimidine derivatives. Cell differentiation was determined by nitroblue tetrazolium-reducing activity, morphologic changes, expression of CD11b, lysozyme activity, and hemoglobin production. RESULTS MS-430 (2-piperidino-5,6-dihydro-7-methyl-6-oxo (7H) pyrrolo [2,3-d] pyrimidine maleate) effectively induced HL-60 cells into mature granulocytes. MS-430 activated the mitogen-activated protein kinase (MAPK) of the cells before causing granulocytic differentiation. MAPK activation was necessary for MS-430-induced differentiation, because PD98059, an inhibitor of MAPK kinase, suppressed the differentiation induced by MS-430. MS-430 also induced monocytic differentiation of THP-1, P39/Tsu, and P31/Fuj leukemia cells, but did not affect erythroid differentiation of K562 or HEL cells. CONCLUSIONS MS-430 potently induces differentiation of some myelomonocytic leukemia cells. This novel synthesized pyrimidine compound shows promise as a therapeutic agent for treatment of leukemia and as a neurotrophic drug.