A novel ATP regeneration system using polyphosphate-AMP phosphotransferase and polyphosphate kinase.

Polyphosphate-AMP phosphotransferase (PAP) and polyphosphate kinase (PPK) were used for designing a novel ATP regeneration system, named the PAP-PPK ATP regeneration system. PAP is an enzyme that catalyzes the phospho-conversion of AMP to ADP, and PPK catalyzes ATP formation from ADP. Both enzymes use inorganic polyphosphate [poly(P)] as a phosphate donor. In the PAP-PPK ATP regeneration system, ATP was continuously synthesized from AMP by the coupling reaction of PAP and PPK using poly(P). Poly(P) is a cheap material compared to acetyl phosphate, phosphoenol pyruvate and creatine phosphate, which are phosphate donors used for conventional ATP regeneration systems. To achieve efficient synthesis of ATP from AMP, an excessive amount of poly(P) should be added to the reaction solution because both PAP and PPK consume poly(P) as a phosphate donor. Using this ATP generation reaction, we constructed the PAP-PPK ATP regeneration system with acetyl-CoA synthase and succeeded in synthesizing acetyl-CoA from CoA, acetate and AMP. Since too much poly(P) may chelate MG2+ and inhibit enzyme activity, the Mg2+ concentration was optimized to 24 mM in the presence of 30 mM poly(P) in the reaction. In this reaction, ATP was regenerated 39.8 times from AMP, and 99.5% of CoA was converted to acetyl-CoA. In addition, since the PAP-PPK ATP regeneration system can regenerate GTP from GMP, it could also be used as a GTP regeneration system.

The gene for an exopolyphosphatase of Pseudomonas aeruginosa.

In Pseudomonas aeriginosa, a gene, ppx, that encodes exopolyphosphatase [exopoly(P)ase; EC 3.6.1.11] of 506 amino acids (56,419 Da) was found downstream of the gene for polyphosphate kinase, ppk. Since ppx is located in the opposite direction of the ppk gene, they do not constitute an operon. The predicted amino acid sequence of PPX is 41% identical with Escherichia coli PPX. The gene product of ppx (paPPX) was overproduced in E. coli, and its activity was evaluated. Orthophosphate (Pi) is released from polyphosphate [poly(P)], the average chain lengths of which are 79 and 750, respectively. The amount of Pi released matched the amount of poly(P) lost. Thus ppx encodes an enzyme that has exopoly(P)ase activity.

The polyphosphate kinase gene of Pseudomonas aeruginosa.

We have cloned and sequenced a gene encoding polyphosphate kinase (PPK) from Pseudomonas aeruginosa PAO1. The gene immediately follows the hemB gene encoding porphobilinogen synthase responsible for heme synthesis. The predicted amino acid sequence of P. aeruginosa PPK is similar to those of PPKs previously characterized except that it possesses an extra stretch of 46 amino acids at its N-terminus, which has significant similarity to the Ras-related protein ARA5 of Arabidopsis thaliana. When P. aeruginosa PPK was overproduced in Escherichia coli, ATP-dependent polyphosphate-synthesizing activity was drastically enhanced, confirming that the protein is a PPK.

Calcium-sensing receptor in mature osteoclasts, which are bone resorbing cells.

Bone metabolism consists of osteoblast-mediated bone formation coupled to osteoclastic resorption of bone. Osteoclastic bone resorption plays an important role in normal skeletal development and the maintenance of its integrity throughout life. Although osteoclastic activity is thought to be under the control of feedback regulation by extracellular cations, the molecular mechanism of detecting extracellular cations within the bone microenvironment remains to be clarified. In the present study we showed by reverse transcription-polymerase chain reaction and Northern blot analysis that cultured mature osteoclasts express the calcium-sensing receptor (CaSR) mRNA. The nucleotide sequence of rabbit osteoclast CaSR was approximately 90% identical to that of CaSR cDNA from human, bovine, and rat parathyroid glands. Moreover, the activity of osteoclastic bone resorption, as determined by pit formation, was regulated by extracellular calcium ion as well as its agonists that are known to act through the CaSR. We conclude that CaSR, homologous to that identified in parathyroid glands, is present in mature osteoclasts and calcium ion released from bone may directly regulate osteoclastic bone resorption.

Inorganic polyphosphate and the induction of rpoS expression.

Inorganic polyphosphate [poly(P)] levels in Escherichia coli were reduced to barely detectable concentrations by expression of the plasmid-borne gene for a potent yeast exopolyphosphatase [poly(P)ase]. As a consequence, resistance to H2O2 was greatly diminished, particularly in katG (catalase HPI) mutants, implying a major role for the other catalase, the stationary-phase KatE (HPII), which is rpoS dependent. Resistance was restored to wild-type levels by complementation with plasmids expressing ppk, the gene for PPK [the polyphosphate kinase that generates poly(P)]. Induction of expression of both katE and rpoS (the stationary-phase sigma factor) was prevented in cells in which the poly(P)ase was overproduced. Inasmuch as this inhibition by poly(P)ase did not affect the levels of the stringent-response guanosine nucleotides (pppGpp and ppGpp) and in view of the capacity of additional rpoS expression to suppress the poly(P)ase inhibition of katE expression, a role is proposed for poly(P) in inducing the expression of rpoS.

Estrogen inhibits bone resorption by directly inducing apoptosis of the bone-resorbing osteoclasts.

Estrogen deficiency causes bone loss, which can be prevented by estrogen replacement therapy. Using a recently developed technique for isolation of highly purified mammalian osteoclasts, we showed that 17 beta-estradiol (E2) was able to directly inhibit osteoclastic bone resorption. At concentrations effective for inhibiting bone resorption, E2 also directly induced osteoclast apoptosis in a dose- and time-dependent manner. ICI164,384 and tamoxifen, as pure and partial antagonists, respectively, completely or partially blocked the effect of E2 on both inhibition of osteoclastic bone resorption and induction of osteoclast apoptosis. These data suggest that the protective effects of estrogen against postmenopausal osteoporosis are mediated in part by the direct induction of apoptosis of the bone-resorbing osteoclasts by an estrogen receptor- mediated mechanism.

Enantioselective fluorination of organic molecules. I. Synthetic studies of the agents for electrophilic, enantioselective fluorination of carbanions.

In order to develop novel methods for electrophilic and enantioselective fluorination of active methine compounds, preliminary experiments were carried out. The N-tosyl derivative 5 obtained from D-phenylglycine was fluorinated with FClO3 or diluted F2 gas to give the N-fluoro-N-tosyl derivative 6. N-tosyl- or N-mesyl-(S)-alpha-phenethylamine 7 or 8 was subjected to FClO3 fluorination to produce the corresponding N-fluoro derivative, 10 or 11, respectively. Enantioselective fluorination of some methine compounds was attempted employing the above N-fluoro agents. Best result was obtained when 2-benzyl-1-tetralone/KHMDS was treated with 10 to produce the fluorinated tetralone 17 in 53% yield with enantiomeric excess (ee) of 48%.

Effect of cyclic GMP produced by natriuretic peptides on osteoblast-like MC3T3-E1 cells.

The C-type natriuretic peptide (10(-7) M) and atrial natriuretic peptide (10(-7) M) enhanced cGMP accumulation by 418 and 83 times the control value, respectively, in osteoblast-like MC3T3-E1 cells. The natriuretic peptide B receptor was assumed to be the major natriuretic peptide receptor. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) activated alkaline phosphatase doubled the activity versus the control value on day 15. Phosphodiesterase activity was not stimulated by the addition of cGMP (1 MicroM). cGMP-dependent protein kinase (G kinase) activity of the supernatant fraction was 25.5 pmol/min/mg protein. The 42 kDa protein band was detected to be phosphorylated by G kinase on SDS-PAGE. These results supported the hypothesis that natriuretic peptides regulate the differentiation of MC3T3-E1 cells through a cGMP-dependent pathway.

Vitamin K2 inhibits osteoclastic bone resorption by inducing osteoclast apoptosis.

In contrast to vitamin K1(VK1), vitamin K2(VK2) inhibited osteoclastic bone resorption by unfractionated bone cells and isolated osteoclasts. To investigate the mechanism of inhibition of osteoclastic bone resorption by VK2, we examined the effect of this vitamin on osteoclast apoptosis using a DNA-binding fluorescent dye, Hoechst 33258. In unfractionated bone cells and isolated osteoclasts on dentin slices, we first demonstrated that VK2 induced osteoclast apoptosis, but VK1 did not. Moreover, cycloheximide inhibited VK2-induced osteoclast apoptosis. These results suggest the possibility that VK2 inhibits osteoclastic bone resorption by targeting osteoclasts to undergo apoptosis, which leads to cell death.

Interleukin-2 receptor positive T and B cells in children with acute severe asthmatic attack.

Subpopulations of interleukin-2 receptor (IL-2R)-positive CD4 and CD8 T cells and IL-2R+CD20 B cells in the peripheral blood lymphocytes as well as serum concentrations of soluble IL-2R (sIL2R) were measured in children aged 1-7 years who suffered acute severe asthmatic attack. Subpopulations of CD4+IL-2R+ cells, CD8+IL-2R+ cells and CD20+IL-2R+ cells in the peripheral blood lymphocytes at acute severe asthmatic attack phase were significantly higher than those at non-asthmatic attack phase (P < 0.02, P < 0.03 and P < 0.02, respectively). Subpopulations of CD20+IL-2R+ cells in the peripheral blood lymphocytes significantly decreased 5-10 days after acute severe asthmatic attack (at recovery phase, P < 0.02) and were significantly correlated with clinical severity of asthmatic attack (P < 0.05). These results indicated that activation of both T cells and B cells was important in the pathogenesis of acute asthmatic attack in young children.

Lymphocyte subpopulations and function in childhood IgA nephropathy.

In order to examine T lymphocyte function in childhood IgA nephropathy, 13 patients and 10 age-matched control subjects were studied. T lymphocyte function was examined in terms of in vitro immunoglobulin synthesis by peripheral blood mononuclear cells (PBMC) and CD4-depleted (suppressor-rich) and CD8-depleted (helper-rich) PBMC in both unstimulated and pokeweed mitogen (PWM) and Staphylococcus aureus Cowan I (SAC) stimulated cultures. T lymphocyte subpopulations were examined by two-color immunofluorescence analysis using Fluorescein-Activated Cell Sorter (FACS). Children with IgA nephropathy showed (1) a significant increase in IgA synthesis by PBMC with or without mitogen stimulation, (2) a significant increase in IgG and IgA synthesis by CD4-depleted (suppressor-rich) PBMC, (3) a significant increase in IgG and IgA synthesis by CD8-depleted (helper-rich) PBMC, and (4) a significant decrease in suppressor-inducer T cells (Leu3a+Leu8+). These results suggest that a decrease in suppressor-inducer T cells, impaired suppressor T cell function and hyperactivity of helper T cell function are responsible for the increase in IgA production in children with IgA nephropathy.

The effect of site and mode of expression of a heterologous antigenic determinant on the properties of poliovirus hybrids.

The antigenic site normally situated in the EF loop of VP2 (2EF) of poliovirus type 2 (Lansing) [PV2 (L)] was expressed in 2EF or in the BC loop of VP1 (1BC) of PV1 (Mahoney) [PV1 (M)]. A hybrid virus expressing the site in 2EF of PV1 (M) is known to be neutralizable by PV2 (L)-specific antisera and to induce neutralizing antibodies against PV-2 (L). In contrast, a hybrid expressing a related sequence in 1BC of PV1 (M), which maintained the length of the native BC loop, was not neutralizable by PV2 (L)-specific antisera and did not induce PV2 (L)-neutralizing antibodies. However, when 1BC was extended, so that it was longer than the native 1BC, the resulting hybrids induced low titers of PV2 (L)-neutralizing antibody although they were still not neutralizable by PV2 (L)-specific antisera. Synthetic peptides copying the extended sequences raised neutralizing antibodies which were able to distinguish between the sequence expressed in the extended 1BC, in the native-length 1BC and in 2EF. The growth rates of the hybrids with modifications to 1BC depended upon the nature of the modifications. The hybrid with the native length 1BC grew poorly, but extending 1BC tended to improve the growth rate, and extending 1BC with PV1 (M)-specific sequences nearly restored wild-type growth rates. Thus, the size, precise sequence and location of a heterologous antigenic site expressed on poliovirus have significant effects on the properties of that determinant and of the hybrid expressing it. Antigenicity of hybrids can be modified and their growth rate can be enhanced by appropriate choices of these parameters.

Phenotypic characterization of antigenic hybrids of poliovirus.

Three poliovirus hybrids, modified in neutralization antigenic sites (NAgs) I or II, were characterized for several phenotypic traits. The modifications to the capsid interfered with some stage of the life-cycle of the virus, since all three hybrids were growth-impaired in comparison to poliovirus type 1 (Mahoney) [PV1 (M)], the wild-type parent virus. All hybrids exhibited a reduced growth rate and a small-plaque phenotype, but they were not temperature sensitive. Furthermore, only one hybrid was slightly less stable to heating than the parent virus; the other two were as stable as the parent. Therefore, decreased thermal stability of the capsid is not an important cause of the poor growth characteristics of these hybrids.

Effects of cytokines on human thymic epithelial cells in culture. II. Recombinant IL 1 stimulates thymic epithelial cells to produce IL6 and GM-CSF.

Our earlier study reported the ability of interleukin 1 (IL1) to promote proliferation and to induce morphological changes of human thymic epithelial cells (TEC) in culture. The present study was undertaken to examine the effects of IL1 on the secretory function of TEC. Both human recombinant IL1 alpha and IL1 beta induced TEC to produce molecules in the culture supernatant fluids (TES) which displayed marked thymocyte proliferative capacities. This activity was specifically induced by IL1 since other TEC growth factors such as epidermal growth factor and a bovine pituitary extract had no effect on promoting secretion of T cell-activating molecules by TEC. Using specific radioimmunoassays for both forms of IL1, we found that unstimulated TEC produced negligible amounts of IL1 alpha and IL1 beta in TES, which were not increased by IL1 stimulation, and we concluded that the IL1-induced TES molecules were not IL1. IL1 induced TEC to produce IL6, as detected by the hybridoma growth factor biological activity. Neutralizing anti-IL6 antibodies completely blocked the thymocyte activating capacities of the IL1-induced TES thus implying a major role for IL6 in TEC-derived T cell activation. IL1 also induced TEC to produce GM-CSF as measured by bioassay and confirmed by an immunoenzymetric assay. Our results confirm that TEC are a source of cytokines and show that TEC respond to IL1 by producing cytokines with consequences on the thymic lymphoid population. This further emphasizes the importance and complexity of paracrine molecular interactions involved in intrathymic development.

[An X-ray crystallographic study on effect of acid etching method to enamel and enhancement of crystallinity of the etched enamel with gel method].

With the purpose of studying the influence of enamel acid etching technique, and the effectiveness of a strengthening of tooth enamel by silicahydro gel method, the following groups were compared, analyzed and observed by the measurements of diffracted X-ray from thin layer specimens and by microarea X-ray diffraction from an X-ray crystallographic point of view. Group I: non-treated tooth enamel Group II: acid-etched tooth enamel Group III: tooth enamel by application of silicahydro gel method after being acid etched. The results obtained were as follows: 1. Qualitative analyses by the measurements of diffracted X-ray from thin layer specimens 1) Ca5(PO4)3F, Ca5F (PO4)3 and Ca3(PO4)2.xH2O were detected in every group, and Ca3(PO4)2.xH2O demonstrated a high reliable value in group III. 2) In comparison with groups I and II, group III clearly revealed a peak shift toward high angle side, however, a halo peak was not recognized in every group. 3) As a result of evaluating crystallinity, crystallinity became favorable in order of group II, group I and group III. 4) A-axis lattice constant shortened in order of group II greater than group I greater than group III, and c-axis lattice constant shortened in order of group II greater than group I = group III. 2. Qualitative analyses by microarea X-ray diffraction 1) Solid solution of Ca5(PO4)3F and Ca5(PO4)3(OH) proved to exist in every group and in each microarea. 2) A halo peak appeared in group II and showed the trend of disappearance in group III, however, no peak shift was observed in all groups. From the foregoing results, the loss of the utmost enamel surface layer exhibiting high crystallinity and the lowering of crystallinity by acid etching technique were revealed from an X-ray crystallographic point of view and furthermore if silicahydro gel method was applied, it was suggested that enamel would be restored or that crystallinity would be enhanced.

Repeat renal biopsy in children with IgA nephropathy.

Serial renal biopsy findings in 61 children with IgA nephropathy were correlated with their clinical course. At the time of the second biopsy, 23 patients showed clinical remission defined as complete disappearance of proteinuria and hematuria with normal renal function while 38 had persistent urinary abnormalities with normal renal function at the second biopsy. There were no differences between the two groups with regard to initial clinical findings and pathologic findings of the initial renal biopsy. The second biopsy of patients with clinical remission showed improvement of the glomerular changes on light microscopy, disappearance or diminution of IgA deposits in the mesangium and decrease of electron-dense deposits, whereas the second biopsy of patients with persistent urinary abnormalities showed progression of glomerular changes on light microscopy, persistence of mesangial IgA deposits and persistence of electron-dense deposits. Our study results show the importance of repeat renal biopsy in children with IgA nephropathy with persistent urinary abnormalities, as a progression of glomerular changes is common in these patients. These observations suggest that the deposition of IgA in the mesangium may be responsible for the glomerular damage in children with IgA nephropathy.

The bond strength of a glass polyalkenoate cement to enamel and stainless steel.

An in vitro evaluation of bond strength of a glass polyalkenoate cement to human dental enamel and stainless steel is reported. Shear testing of adhesive strength was measured over time at 23 degrees C following a one minute mixing time. It was found that there is a significant difference in bond strengths between enamel and stainless steel with strength to enamel the greater. Bond strength increases with time with very low strength measured within 30 minutes from commencement of mixing.

[Study on the formation of fluorapatite with freeze-drying silicahydro gel method].

Resultant products and gel substance, formed with silicahydro gel method as well as with freeze-drying silicahydro gel method by use of tray, were compared and examined to materialize clinical application of gel method easily and the following results were obtained: 1. Evaluation of gel substance in control group gel (silicahydro gel) and in experimental group gel (gel prepared by the addition of deionized water to freeze-drying silicahydro gel) As the results of measuring the estimation of F- by ion chromatography and pH value, no clear-cut distinction between control group gel and experimental group gel was recognized, however, it was implied by naked-eye observations and through scanning electron microscope with the passage of time that experimental group gel had been inferior to control group gel. 2. Evaluation of resultant products in control group gel (resultant products formed with silicahydro gel method) and in experimental group gel (resultant products formed with freeze-drying silicahydro gel method) Naked-eye observations of reacting progress, the pH measurements, the calculation of Ca/P ratio, qualitative analyses by X-ray diffraction (reference; the measurements of crystallite size, lattice imperfection and lattice constant) and composition analyses by infrared absorption spectrum were carried out and as the results, resultant products in control group gel proved to be more favorable than those in experimental group gel. From the foregoing outcomes, it was suggested that fluorapatite of favorable crystallinity had been possible to be produced more with silicahydro gel method than with freeze-drying silicahydro gel method, and that further investigation inclusive of gel substance would be required in the event of clinically applying freeze-drying silicahydro gel method effectively.

[Study on the physico-chemical properties of fluoride gel in application of freeze-drying gelatin gel method (tray method)].

As a means of studies of securing freeze-drying method by application of tray for prevention of white spots, decalcification and secondary caries of enamel, which may develop during and after orthodontic treatment, comparison and review were made on physico-chemical properties of fluoride gel to be used for gelatin gel method and freeze-drying gelatin gel method (control group gel and experimental group gel), and the following results were obtained: 1. Physical properties As the results of naked-eye observations, morphological observations through scanning electron microscope and measurements of gelly strength and viscosity, no distinct difference between control and experimental group gel was noted. 2. Chemical properties The qualitative analyses of fluoride ion with ion chromatogram and pH measurement were carried out, and consequently experimental group gel indicated similar values to control group gel. From the foregoing results, it was learned that no significant difference in their physico-chemical properties was clearly confirmed between control and experimental group gel, and it was suggested that similarly to control group gel, experimental group gel would be an effective gel substance as gel media for the formation process of fluorapatite or the reinforcement method of tooth enamel.

[Study on frictional characteristics of KB horizontal brackets. A comparative study of kinetic frictional forces to be caused between various kinds of brackets and wires].

KB horizontal brackets were designed to tip no more than 6 degrees at the maximum. This tipping amount is based on the idea of reducing friction between a wire and brackets to allow the effective tooth movement of the Begg technique even with horizontally long brackets, and does not originate in the concept of carrying out tipping movement. Thereon, experimental measurements by use of Rheometer were conducted to review for comparison of the kinetic frictional forces caused between various wires and the following four types of brackets; KB horizontal brackets, Tip edge brackets, Straight edge brackets and Begg brackets. 1. In case of utilizing ribbon arch wires and rectangular wires, no significant difference was acknowledged among Tip edge, KB horizontal and Straight edge brackets. 2. There proved to be a reduction in the kinetic frictional forces by incorporating tip into the edgewise slots, when using smaller dimensions of the wires which call for the effective tooth movement, however, Begg brackets (in conjunction with Ordinary T-pins and/or Safety T-pins) showed the small value which is far less than that of the three kinds of brackets.