Behaviour and adsorptive removal of siloxanes in sewage sludge biogas.

We investigated the behaviour of siloxanes, which adversely affect biogas engines, as well as their concentration levels in sewage sludge biogas in Japan. We also performed experiments on the absorptive removal of siloxanes using various adsorbents and determined the main adsorbent characteristics required for the removal of siloxanes. The results of our study on the concentration and composition of siloxanes in biogas were similar to previous reports. Moreover, we found that the concentration of siloxanes changes in relation to the outside air temperature based on real-time measurements of siloxanes using a continuous analyser. We further speculated that the continuous analyser would accurately indicate the siloxane concentration in model biogas but overestimate the siloxane concentration in actual biogas because of positive interference by VOCs and other biogas components. In the siloxane adsorption experiment, the equilibrium uptake of both cyclic siloxanes, D4 and D5, was positively related to the BET-specific surface area of the adsorbents and the fraction of the external surface area taken up by relatively large diameter pores. We attributed the adsorption results to the fact that the siloxane molecules are generally larger than micropores; therefore, they are less susceptible to adsorption to micropores. Based on these results, we concluded that adsorbents with large BET-specific surface areas, especially those with a high external specific surface area and pores of relatively large diameters, are desired for the removal of siloxanes.

Refolding of firefly luciferase immobilized on agarose beads.

The renaturation yield of the denatured firefly luciferase decreased strongly with increasing protein concentration in a renaturation buffer, because of aggregation. In this study, firefly luciferase was immobilized on agarose beads at a high concentration. Although the protein concentration was extremely high (about 100-fold) compared to that of soluble luciferase, the renaturation yield was comparable with that for the soluble one. Thus, immobilization was shown to be effective for avoiding aggregation of firefly luciferase. It was also shown that the optimum buffer conditions for renaturation of the immobilized luciferase were the same as those for the renaturation in solution. Also, it was indicated that electrostatic interactions between a protein and the matrix have a negative effect on renaturation of the immobilized luciferase since the renaturation yield decreased at acidic pH only for the immobilized luciferase. These novel observations are described in detail in this paper.

Monitoring of the refolding process for immobilized firefly luciferase with a biosensor based on surface plasmon resonance.

In order to examine the possibility of the use of a surface plasmon resonance (SPR) sensor for real-time monitoring of the process of refolding of immobilized proteins, the refolding of firefly luciferase immobilized on a carboxymethyldextran matrix layer was analyzed. The SPR signal of the immobilized luciferase decreased after unfolding induced by GdnCl and increased gradually in the refolding buffer, while there was no signal change in the reference surface lacking the immobilized protein. The decrease in the SPR signal on unfolding was consistent with the difference between the refractive indices of the native and unfolded protein solutions. The effects of blocking of the excess NHS-groups of the matrix layer on the refolding yield were examined by means of an SPR sensor. The results were consistent with those obtained with the enzymatic activity assay, indicating that the changes in the SPR signal reflected the real-time conformational changes of the immobilized protein. Hence, an SPR biosensor might be used for monitoring of the process of refolding of immobilized proteins and as a novel tool for optimization of the refolding conditions. This is the first demonstration that SPR signal changes reflect the conformational changes of an immobilized protein upon unfolding and refolding.

Roles of glutamate receptor subtypes in the development of vestibular compensation after unilateral labyrinthectomy in the guinea pig.

We investigated the roles of ionotropic glutamate receptor subtypes in the development and recovery of spontaneous nystagmus (SN) after unilateral labyrinthectomy (UL) in guinea pigs. When administered at 3 h after UL, N-methyl-D-aspartate (NMDA) and kainate (KA), which are NMDA and non-NMDA receptor agonists, respectively, increased the frequency of SN. The effect of KA was more potent than that of NMDA. In contrast to these agonists, MK-801 and CNQX decreased the frequency of SN. Although the administration of KA at 48 h after UL increased the frequency of SN, it did not exhibit any effects at 72 h after UL. MK-801 caused a recurrence of SN following administration at 48 and 72 h after UL. Neither NMDA nor CNQX exhibited any effects after administration at 48 or 72 h after UL. A newly synthesized compound, NC-1200, which has inhibitory action on the glutamate response, decreased the frequency of SN in a dose-dependent manner following administration at 3 h after UL, but did not exhibit any effects when administered at 48 and 72 h after UL. From these results, it was found that NMDA and non-NMDA receptors play important roles in the development of SN after UL, and that the NMDA receptor contributes to the development of ocular motor compensation.

Ribosomal RNA supplementation highly reinforced cell-free translation activity of wheat germ.

We have constructed an inexpensive, highly efficient eukaryotic cell-free translation system. Wheat germ rRNA (WG rRNA) was prepared by phenol/chloroform (P/C) extraction, a simple and quick method, from wheat germ, an inexpensive and commercially available by-product of flour production. Addition of a small amount of WG rRNA into a wheat germ cell-free translation system increased the protein productivity of the system 6- to 8-fold. Isolated 18S or 28S rRNA alone enhanced the protein production only 2-fold or 3.9-fold, respectively, at maximum. On the other hand, their equimolar mixture enhanced the production as much as the whole WG rRNA, indicating 18S and 28S rRNA synergistically functioned to enhance protein synthesis. Addition of WG rRNA slightly improved the stability of mRNA in the cell-free translation system, which explained only partly the enhancement of protein production. Addition of WGE or ribosome containing approximately the same amount of rRNA in the form of protein-rRNA complex as WG rRNA added to the system did not increase the protein production in the translation system. When ribosome in the cell-free translation system was replaced with WG rRNA, the system did not exhibit any detectable translation activity, indicating that the translation activity of WG rRNA is negligible in comparison with that of ribosome. These results indicated that WG rRNA affected some mechanisms regulating the translation rate in wheat germ cell-free system, resulting in increased protein production.

Microbial ecology of nitrifying bacteria in wastewater treatment process examined by fluorescence in situ hybridization.

The microbial ecology of nitrifying bacteria in various types of wastewater treatment processes and the dynamic response of the microbial ecology in biofilms were investigated using fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. Nitrifying bacteria were found to exhibit various organizational forms under different conditions of substrate composition and concentration. Ammonia-oxidizing bacteria were dominant in ammonia-rich inorganic wastewater, while heterotrophic bacteria and ammonia-oxidizing bacteria were localized at different positions in the biofilm in organic wastewater. The dynamics of the microbial ecology in the biofilm with regard to the spatial distribution of ammonia-oxidizing bacteria and heterotrophic bacteria caused by a gradual change in substrate composition was successfully monitored by FISH analysis.

Bone mineral measurement in Japan.

Various methods for evaluating bone mineral in appendicular, and axial bone or in the whole skeleton have recently become available. As bone mineral is one of the major determinants of bone strength, its exact measurement should be useful for the diagnosis of osteoporosis, as well as for the prediction of fracture risk and monitoring of therapeutical response. The aims of this paper are to review the fundamental performance of bone mineral measurements, the improvements in DXA systems, and the progress in site-specific bone mineral instruments for the radius and calcaneus used in Japan, and to introduce diagnostic criteria for primary osteoporosis, and report on annual rates of bone loss in Japanese females.

Real-time monitoring of ammonia-oxidizing activity in a nitrifying biofilm by amoA mRNA analysis.

Ammonia monooxygenase encoding mRNA (amoA mRNA) transcription in the wastewater treatment process was investigated using reverse transcription PCR (RT-PCR) as the model indicating specific function and activity in nitrifying processes. The dynamic response of amoA mRNA transcription and ammonia-oxidizing activity to the change of environmental conditions such as pH and concentration of ammonia was examined to determine the inductive factor and the inhibitor for amoA mRNA expression. Furthermore, we semiquantitatively investigated the response of amoA mRNA transcription to the pH fluctuation in a continuous fed nitrifying reactor. As a result, amoA mRNA oriented analysis enabled real-time assay of ammonia-oxidizing activity within 2 h as a response time. In contrast, rRNA and amoA encoding DNA were constantly detected at almost the same amount throughout the experiment. mRNA transcription was regulated by the many environmental conditions: ammonia seems to be one of the strong inducers for transcription of amoA mRNA, whereas low pH seems to be a strong inhibitor. These factors simultaneously affected the mRNA transcription and enzymatic activity leading to the complex phenomena of ammonia-oxidizing activity and amoA mRNA transcription in the continuous feeding reactors.

[Detection of rubella virus antibody by enzyme linked immunosorbent assay with parallel line method].

An attempt was made to measure the antibody to rubella virus in human sera by ELISA by calculating the relative potency by the method of the parallel line assay. As a standard serum, adult serum 8 months after rubella infection was used. Sample sera were collected from 42 infants before MMR vaccination and 8-10 weeks after the vaccination and 48 adults three years or more after rubella vaccination. In addition, three infant sera with rubella 6-19 days after onset of rash and three gamma immunoglobulin products were used. The dose response curve of the standard serum and that of the sample serum showed the lineality and the regression line of the sample serum was parallel to that of the standard serum. And the relative potency of sample sera was successfully calculated. The good correlation was observed between the rubella antibody titer by hemagglutination inhibition test and that measured by ELISA. The coefficients of variance of relative potency obtained from four repeated measurements for two weeks showed from 15% to 25% in four sera of HI high titer. The reproducibility of parallel line assay was also satisfactory.

[Microtiter enzyme-linked immunosorbent assay for diphtheria antitoxin in human sera--application of parallel line assay method].

The antibodies against Diphtheria toxin in human sera were measured by the ELISA method and the antitoxin titer was calculated using the parallel line assay method which is a quantitative bioassay method. The reproducibility of this method and the comparison of the titer between this method and cell culture method were proven. The dose response curve of standard antiserum and test serum calculated by the parallel line assay method showed linearity, and the regression line of the test serum was parallel to the standard serum. The coefficients of variance (CV) of the antitoxin titers obtained from triplicate measurements of 3 serum samples ranged from 9.1 to 36.0%. The serum at low titer gave a higher CV level. A good correlation was observed between the Diphtheria antitoxin titer by the neutralizing test in cultured cells and that measured by ELISA by the parallel line assay method. The coefficient of regression was 0.996 and the coefficient of the correlation was 0.899.

[In vitro and in vivo activities of cefpodoxime proxetil against penicillin-resistant Streptococcus pneumoniae].

We evaluated in vitro and in vivo activities of cefpodoxime proxetil (CPDX-PR) in comparison with other oral beta-lactams, cefdinir (CFDN), cefditoren pivoxil (CDTR-PI), and faropenem (FRPM), against penicillin-susceptible and -resistant Streptococcus pneumoniae. In vitro activities (MICs) of CPDX, CFDN, CDTR, and FRPM against clinical isolates, penicillin-susceptible S. pneumoniae (PSSP: MIC of penicillin G, < or = 0.063 microgram/ml), penicillin-intermediate S. pneumoniae (PISP: MIC of penicillin G, 0.125-1 microgram/ml), and penicillin-resistant S. pneumoniae (PRSP: MIC of penicillin G, > or = 2 micrograms/ml), were tested by an agar dilution method. The MIC80s of CPDX against 27 PSSP strains, 23 PISP strains, and 23 PRSP strains were 0.032, 1, and 8 micrograms/ml, respectively, which were superior to or equal to those of CFDN (0.063, 4, and 8 micrograms/ml) and were inferior to those of CDTR (0.016, 0.5, and 1 microgram/ml) and FRPM (< or = 0.008, 0.25, and 1 microgram/ml). Infection was induced in mice by inoculating with a PRSP clinical isolate, 9605 or 9601 (serotype 6), or 10692 (serotype 19), through the nares of male ddY mice into the lungs. The mice were treated with drugs with doses of 2-50 mg/kg at 18, 26, 42, and 50 hours after the infection. Viable cell numbers in the lungs and blood were assayed at 66 hours after the infection. The efficacy of each drug was dose-dependent. CPDX-PR showed the most potent in vivo efficacy among the drugs tested against the infections caused by the PRSP strains. MICs of the drugs against PRSP 9605, 9601, and 10692 were as follows: CPDX, 4, 4 and 2 micrograms/ml; CFDN, 16, 16, and 4 micrograms/ml; CDTR, 1, 1, and 0.5 microgram/ml; and FRPM, 1, 0.5, and 0.5 microgram/ml, respectively. Thus, CPDX-PR showed a stronger in vivo activity than that expected from the MICs of CPDX. This was probably caused by the pharmacokinetic advantage of CPDX over the other drugs used in this study.

[Compartment analysis of 123I-iomazenil brain on early and delayed SPECT].

We investigated the characteristics of 123I-Iomazenil (IMZ) SPECT images in 12 adults (six males and six females, with a mean age of 56.1 years). The washout rate of 123I-IMZ from the brain was estimated from two SPECTs done 15 min and 3 hr after injection. Although the washout was relatively slow, the rates differed in each intracerebral region, suggesting that the distribution of 123I-IMZ was gradually changing. Furthermore, assuming 123I-IMZ kinetics in the brain for the three-compartment, two-parameter model, the transition rate constant (K1) from the blood to the brain and the binding potentials (BP) of benzodiazepine to the receptor were calculated. The BP and K1 values were compared with 123I-IMZ SPECT counts and CBF values by 123I-IMP. The BP values correlated more closely with the counts on the delayed SPECT than those on the early SPECT. It was confirmed that delayed SPECT images reflect better the distribution of the benzodiazepine receptor than early images do. On the other hand, the K1 values correlated highly with CBF obtained by 123I-IMP, and this finding suggested that super-early SPECT images might be remarkably influenced by the distribution of CBF.

[Development of a simple method for the washout correction of 123I-IMP SPECT and its application to a quantitative rCBF method].

We have developed a simple method to correct the washout of tracer from the brain based on the two-compartment model in brain early SPECT using N-isopropyl-p-[123I]iodoamphetamine (123I-IMP). This correction was applied to a new quantitative method of regional cerebral blood flow (rCBF) in combination with the microsphere method by continuous arterial sampling previously reported. Data acquisition of 123I-IMP early SPECT was started from 35 min after 123I-IMP i.v. injection, and the time activity curve of whole brain on anterior head planar images was monitored immediately after 123I-IMP i.v. injection for the correction of washout of tracer from the brain. The usefulness of this method was evaluated in 12 patients with various brain diseases by comparison with the results obtained from the super-early SPECT at 7-10 min after 123I-IMP i.v. injection. The washout rates in cases of early SPECT corrected by this method ranged from 16.91% to 39.34% with a mean +/- SD of 27.72 +/- 5.44%. The contrast of hypo- to hyperperfusion regions on early SPECT was improved by the correction of the washout, and its intracerebral distribution was similar to the simultaneously obtained super-early SPECT images. These results indicated that the present correction method for the washout was useful for more correct quantification of rCBF.

Establishment of an apoptosis-suppressible,cell-cycle arrestable cell line and its applicationfor enhancing protein production of serum-free or-supplemented culture.

Expression of c-jun gene induces apoptosis ofcells cultured in serum-free medium. It also promotescell-cycling in serum-containing medium, leading cellsto die by overgrowth. Previously, we established anapoptosis-suppressible, cell-cycle arrestable cellline, c-jun AS, by transfecting Friend murineerythroleukemia (F-MEL) cells with adexamethasone-inducible antisense c-jun gene.Induction of the antisense c-jun transcriptionwith dexamethasone suppressed c-jun expression.As a result, c-jun AS cells survived inserum-free medium containing dexamethasone for a longtime, while F-MEL cells died quickly in the presenceor absence of dexamethasone. In serum-containingmedium, the growth of c-jun AS cells was viablyblocked by inducing antisense c-juntranscription, and the cells survived at thenon-growth state avoiding overgrowth. In the presentstudy, protein productivity of c-jun AS cellswas examined in comparison with that of wild typeF-MEL cells. C-jun AS and F-MEL cells werefurther transfected with a vector for expressingalkaline phosphatase as a protein to be produced, andnamed c-jun AS-SEAP and F-MEL-SEAP cells,respectively. In the serum-free medium withdexamethasone, c-jun AS-SEAP cells produced theprotein for up to 6 days, while F-MEL-SEAP cellsstopped production on day 3 due to cell death causedby serum deprivation. In the serum-containing mediumwith dexamethasone, c-jun AS-SEAP cells wereviably arrested in the cell cycle, and cell death dueto overgrowth was avoided. As the result, they couldproduce the protein for up to 18 days, whileF-MEL-SEAP cells stopped production within 7 days dueto cell death caused by overgrowth.

Reinforcing apoptosis-resistance of COS and myeloma cells by transfecting with bcl-2 gene.

COS, myeloma and HeLa cells, which are commonly used for protein production by cell culture, were transfected with human bcl-2 gene encoded on the shuttle vector BCMGS. Expression of human bcl-2 improved survival of cells remarkably, mildly, or negligibly for COS, myeloma, and HeLa, respectively. Four clones were obtained from the human bcl-2 expressing cell population of COS cells. They expressed human bcl-2 almost at the same level. The viable cell numbers were 6, 2.5, 2.5, and 0.8 times as many for the clones #8, #5, #6, and #7, respectively, as for the control COS cells, when they were cultured at low (0.2%) serum concentration for 9 days. The bcl-2 overexpressing COS cells showed morphology different from that of the control COS cells in serum limited condition. When transfected with mouse lambda protein gene carried by an SV40-derived vector, clone #8 of the bcl-2 transfected COS cells continued the transient expression of lambda protein longer than the control COS cells.

Observation of charge state and conformational change in immobilized protein using surface plasmon resonance sensor.

Behaviors of proteins immobilized on a solid surface were investigated using BIACORE, a biosensor utilizing surface plasmon resonance. This sensor is usually used for analyzing binding events during biomolecular interactions. Here we propose a novel use of this sensor to monitor two kinds of intramolecular changes in immobilized proteins. Several proteins were covalently attached to dextran chains on the sensor surface in the flow cell and were then exposed to a series of buffers with varying pH. Signal changes derived from changes of refractive index around the sensor surface were detected during and after the exposure to each of these buffers, which we denoted as in situ values and postvalues, respectively. The in situ value reflects the behavior of immobilized proteins in these buffers and was revealed to have a correlation with total charge state of the proteins, while the postvalue reflects how immobilized proteins react after the exposure and was suggested to represent the degree of conformational changes of the proteins. This method is expected to be applicable to various analyses and can provide us with new information about the behavior of proteins on solid phase.

Design of the linkers which effectively separate domains of a bifunctional fusion protein.

With the aim of separating the domains of a bifunctional fusion protein, the ability of several lengths of helix-forming peptides to separate two weakly interacting beta-can domains was compared with that of flexible linkers or of a three alpha-helices bundle domain. We introduced helix-forming peptide linkers A(EAAAK)nA (n = 2-5) between two green fluorescent protein variants, EBFP and EGFP, and investigated their spectral properties. The fluorescence resonance energy transfer from EBFP to EGFP decreased as the length of the linkers increased. The circular dichroism spectra analysis suggested that the linkers form an alpha-helix and the alpha-helical contents increased as the length of the linkers increased. The results clearly suggested the ability of the helical linkers to control the distance and reduce the interference between the domains. This 'linker engineering' may open a way to the rational design of linkers which maximize the multiple functions of fusion proteins or de novo multi-domain proteins.

Establishment of an apoptosis-resistant and growth-controllable cell line by transfecting with inducible antisense c-Jun gene.

F-MEL cells were transfected with the c-jun antisense gene located downstream of a glucocorticoid-inducible MMTV promoter, and the obtained cells were named c-jun AS cells. When the c-jun AS cells were treated with dexamethasone (DEX) in DMEM supplemented with 10% serum, the growth of the cells was completely suppressed for a duration of 16 days with a high cell viability exceeding 86%. The c-jun expression in the c-jun AS cells was suppressed moderately in the absence of DEX and strongly in the presence of DEX. The c-jun AS cells grew well and reached a density of 10(6) cells/mL without supplementation of any serum components. Viability was greater than 80% after the cells had been cultured for 8 days in the absence of DEX. The c-jun AS cells stayed at a constant cell density and high viability above 80% for 8 days when they were cultured in the presence of DEX under serum deprivation. In contrast, the wild type F-MEL cells were unable to grow and died by apoptosis in 3 days under serum deprivation. Internucleosomal cleavage of DNA, a landmark of apoptosis, was clearly detectable. Thus the c-jun AS cell line that is resistant to apoptosis induced by serum deprivation and can reversibly and viably be growth-arrested was established. A dual-signal model was proposed to explain the experimental result, the interlinked regulation of apoptosis, and growth by c-jun.

How does heme axial ligand deletion affect the structure and the function of cytochrome b(562)?

We have recently generated a new mutant of cytochrome b(562) (cytb(562)) in which Met7, one of the axial heme ligands, is replaced by Ala (M7A cytb(562)). The M7A cytb(562) can bind heme and the UV-visible absorption spectrum is of a typical high-spin ferric heme. To investigate the effect of the lack of Met7 ligation on the structural integrity of cytb(562), thermal transition analyses of M7A cytb(562) were conducted. From the thermodynamic parameters obtained, it is concluded that the folding of M7A cytb(562) is comparable to the apoprotein despite the presence of heme. On the other hand, exogenous ligands such as cyanide and azide ions are readily bound to the heme iron, indicating that the axial coordination site is available for substrate binding. The peroxidase activity of this mutant is thus examined to evaluate new enzymatic function at this site and M7A cytb(562) was found to catalyze an oxidation reaction of aromatic substrates with hydrogen peroxide. These observations demonstrate that the Met7/His102 bis-ligation to the heme iron is crucial for the stable folding of cytb(562), whereas the functional conversion of cytb(562) is successfully achieved by the loose folding together with the open coordination site.