Tracer experiment and RTD analysis of DAF separator with bar-type baffles.

This paper describes the development of a new dissolved air flotation (DAF) separator with a flow streamlining baffle to improve solid separation efficiency. The analysis of the RTD (residence time distribution) curves indicated that the parameter θ(10) (dimensionless time at which 10% of tracer has discharged) increased from 0.38 for control reactor to 0.54 for the test reactor, suggesting significant reduction in short circuit flow. The RTD curves were also used to develop a compartment model for white water (rich in micro-bubbles and water flow is turbulent) and clear water (little or no air content and water flow is quiescent) zones in the reactor using a series of CSTR (continuous stirred tank reactors) and plug flow regime respectively. The proportion of the volume occupied by the white water zone was different in control and test configurations. In the test reactor, the fraction of the clear water zone was found to increase from 6 to 37%, resulting in improvement of the suspended solid (SS) removal efficiency from 97 to 99%.

Three-dimensional analysis of pulp chambers in maxillary second deciduous molars.

OBJECTIVE: The purpose of this study was to clarify the positional relationship between the crown contour and the pulp chamber using micro-CT system for restorations of maxillary second deciduous molar. METHODS: Five maxillary second deciduous molar teeth each from the deciduous dentition and the mixed dentition periods were used. The positional relationship between the crown contour and pulp chamber was three-dimensionally observed by micro-CT. In addition, the thickness of the dentin at each of the pulp horns and the volume ratio of the pulp chamber against the whole crown were measured. RESULTS: In relationship to the crown contour and the pulp chamber, the pulp horn of the mesiobuccal cusp showed greatest protrusion and eminent swelling of its mesial wall in both the deciduous dentition and the mixed dentition periods. Furthermore, the pulp chamber was shifted to the mesial side. The volume ratio of the pulp chamber to the whole crown, the ratio in the mixed dentition period showed to be significantly smaller than in the deciduous dentition period. The dentin thickness at the mesiobuccal pulp horn was the thinest. CONCLUSIONS: These results suggest that in preparation of cavities in maxillary second deciduous molars, care is necessary not to expose of the mesiobuccal pulp horn. Especially, the mesial wall of the mesiobuccal pulp horn should be noted.

Thymidylate synthase predictive power is overcome by irinotecan combination therapy with S-1 for gastric cancer.

The predictive values of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) gene expressions were retrospectively evaluated in patients with gastric cancer treated by a regimen containing S-1. The study population consisted of 53 patients registered into different two phase II studies for metastatic gastric cancer; 27 patients treated by S-1-alone study: 26 patients treated with S-1 combined with irinotecan (CPT-11). TS and DPD gene expressions in primary tumours were measured by the real-time reverse transcription PCR method. There was no statistical difference in DPD gene expression in terms of response in cases treated with S-1 alone and those treated with S-1 plus CPT-11. TS mRNA of responding tumours was lower than that of nonresponding ones when treated with S-1 (P<0.005). In the S-1-alone group, taking TS cutoff as the median values, the response rate in the low TS group was 50%, but only 8% in the high TS group (P<0.05). Patients with low TS gene expression survived longer than those with high TS gene expression (P<0.0001). However, there was no statistically significant difference in response rate and survival between patients with low TS tumours and those with high TS tumours, when the cutoff was taken as the median value of TS gene expression in the group treated with S-1 plus CPT-11. In conclusion, treatment effects of S-1 monotherapy for gastric cancer were determined by the status of TS gene expression, regardless of DPD gene expression. TS predictive power was overcome by CPT-11 combination therapy with S-1.

Spatial distribution and histogenesis of colorectal Paneth cell metaplasia in idiopathic inflammatory bowel disease.

BACKGROUND AND AIM: Colorectal Paneth cell metaplasia (PCM) is known to be a sign of idiopathic inflammatory bowel disease (IBD), although its distribution and histogenesis are not fully understood. Objectives of this research were to investigate the spatial distribution of PCM in IBD and other forms of colitis (non-IBD), and to find stimuli causing PCM. METHODS: We studied multiple biopsy specimens from 181 patients with ulcerative colitis (UC), 159 with Crohn's disease (CD), 448 with non-IBD, and 78 normal controls. Paneth cell metaplasia frequency, at each colorectal site, was evaluated to find possible differences among diseases, phases of activity, and extents of disease. RESULTS: In non-IBD and controls, PCM was rarely (0-1.9%) seen at distal sites, but frequently (up to 48.7%) found at the ascending colon and cecum (P < 0.001). Paneth cell metaplasia frequency was significantly higher in IBD than in non-IBD patients and controls at distal sites (P < 0.001), but did not differ significantly between UC and CD, or among active, resolving, and quiescent phases. In UC, proctitis and left-sided colitis rarely displayed PCM at unaffected sites. Multiple logistic regression analysis revealed that PCM was positively associated with crypt distortion and mononuclear cell infiltration (P < 0.005), but negatively or not significantly associated with crypt atrophy, mucin depletion, acute inflammation, or phase of activity. CONCLUSIONS: Paneth cell metaplasia is a non-specific phenomenon in the proximal colon, but distal PCM, which occurs exclusively in affected mucosa, is a useful marker indicating IBD, even in the inactive phase. Regression analysis suggests that repair and regeneration may be the most potent stimuli causing PCM.

Carbonyl reductase: a novel metastasis-modulating function.

To explore reasons for differences in the malignancy of tumors, we have compared two cell lines derived from a mouse lung adenocarcinoma cell line that differ 10-fold in their capacity to form lung metastases from s.c. primary tumors or after i.v. injection. One mRNA encoding carbonyl reductase was identified at a relatively high abundance in the subline with low metastatic capacity but was not detectable in the highly metastatic subline. Transfection of the former subline with a plasmid construct expressing antisense carbonyl reductase rendered the cells highly metastatic. Conversely, the capacity of the highly metastatic cells to metastasize was markedly reduced after transfection with a construct expressing carbonyl reductase. We also found that human prostate cancers show loss of carbonyl reductase expression compared with normal prostate epithelia. These data suggest that carbonyl reductase has an important function in modifying the metastatic behavior of malignant tumors.

How do the x-ray structure and the NMR structure of FMN-binding protein differ?

The crystal structure of FMN-binding protein (FMN-bp) from Desulfovibrio vulgaris Miyazaki F was solved by the multiple isomorphous replacement method and refined to an R factor of 15.1% at 1.3 A resolution. FMN-bp exists in a dimeric form in the crystal, in contrast to the monomeric structure determined by NMR. R.m.s. deviations between the crystal structure and the solution structure are more than 2 A, which implies significant differences. There are some hydrophobic residues in the interface between the two monomers. In particular, Leu122 in the C-terminus has a close contact with the o-xylene moiety of FMN, while solvent molecules may cover the o-xylene moiety in the solution structure.

Optical coherence tomography of the neurosensory retina in rhegmatogenous retinal detachment.

PURPOSE: To clarify the pathologic changes of the detached neurosensory retina in rhegmatogenous retinal detachment. METHODS: Retinal images were prospectively examined by optical coherence tomography in 25 eyes of 25 consecutive patients with rhegmatogenous retinal detachment. We excluded the patients whose retinal detachment did not involve the central fovea or patients with poor fixation during optical coherence tomography (OCT) examination. Optical coherence tomography was scanned through the center of the fovea. The patients ranged in age from 15 to 77 years (mean, 45 years; SD, 20 years). The period from onset of subjective symptoms of retinal detachment to OCT ranged from 2 to 60 days (mean, 16 days; SD, 18 days). Optical coherence tomography findings, best-corrected visual acuity, and the height of the retinal detachment at the central fovea were statistically analyzed using ANCOVA (analysis of covariance) and the Mann-Whitney U test. RESULTS: In 25 eyes of 25 patients, OCT of the detached neurosensory retina at and adjacent to the center of the fovea demonstrated normal retinal structure (10 eyes, 40%), intraretinal separation (7 eyes, 28%), and an undulated separated outer retina (8 eyes, 32%). Three statistically significant factors affected best-corrected visual acuity: intraretinal separation (P = .001), intraretinal separation with undulated outer retina (P = .001), and height of retinal detachment at the central fovea (P<.001). Best-corrected visual acuity was significantly worse in the 15 eyes with intraretinal separation with or without an undulated outer retina than in the 10 eyes with retinal thickening but no intraretinal separation (P = .036). The eight eyes with undulated separated outer retina showed significantly higher retinal detachment at the central fovea than the seven eyes with intraretinal separation but no undulated outer retina (P = .009) and the 10 eyes without intraretinal separation (P = .016). The duration from onset of subjective symptoms to OCT was not related to the occurrence of intraretinal separation of the detached retina. CONCLUSIONS: Intraretinal separation of the detached retina occurred frequently and shortly after retinal detachment in rhegmatogenous retinal detachment and was one of the factors associated with poor vision in rhegmatogenous retinal detachment. Best-corrected visual acuity significantly decreased in the highly detached retina.

A double-strand break repair component is essential for S phase completion in fission yeast cell cycling.

Fission yeast rad22(+), a homologue of budding yeast RAD52, encodes a double-strand break repair component, which is dispensable for proliferation. We, however, have recently obtained a cell division cycle mutant with a temperature-sensitive allele of rad22(+), designated rad22-H6, which resulted from a point mutation in the conserved coding sequence leading to one amino acid alteration. We have subsequently isolated rad22(+) and its novel homologue rti1(+) as multicopy suppressors of this mutant. rti1(+) suppresses all the defects of cells lacking rad22(+). Mating type switch-inactive heterothallic cells lacking either rad22(+) or rti1(+) are viable, but those lacking both genes are inviable and arrest proliferation with a cell division cycle phenotype. At the nonpermissive temperature, a synchronous culture of rad22-H6 cells performs DNA synthesis without delay and arrests with chromosomes seemingly intact and replication completed and with a high level of tyrosine-phosphorylated Cdc2. However, rad22-H6 cells show a typical S phase arrest phenotype if combined with the rad1-1 checkpoint mutation. rad22(+) genetically interacts with rad11(+), which encodes the large subunit of replication protein A. Deletion of rad22(+)/rti1(+) or the presence of rad22-H6 mutation decreases the restriction temperature of rad11-A1 cells by 4-6 degrees C and leads to cell cycle arrest with chromosomes incompletely replicated. Thus, in fission yeast a double-strand break repair component is required for a certain step of chromosome replication unlinked to repair, partly via interacting with replication protein A.

A new mode of B12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase.

BACKGROUND: Diol dehydratase is an enzyme that catalyzes the adenosylcobalamin (coenzyme B12) dependent conversion of 1,2-diols to the corresponding aldehydes. The reaction initiated by homolytic cleavage of the cobalt-carbon bond of the coenzyme proceeds by a radical mechanism. The enzyme is an alpha2beta2gamma2 heterooligomer and has an absolute requirement for a potassium ion for catalytic activity. The crystal structure analysis of a diol dehydratase-cyanocobalamin complex was carried out in order to help understand the mechanism of action of this enzyme. RESULTS: The three-dimensional structure of diol dehydratase in complex with cyanocobalamin was determined at 2.2 A resolution. The enzyme exists as a dimer of heterotrimers (alphabetagamma)2. The cobalamin molecule is bound between the alpha and beta subunits in the 'base-on' mode, that is, 5,6-dimethylbenzimidazole of the nucleotide moiety coordinates to the cobalt atom in the lower axial position. The alpha subunit includes a (beta/alpha)8 barrel. The substrate, 1,2-propanediol, and an essential potassium ion are deeply buried inside the barrel. The two hydroxyl groups of the substrate coordinate directly to the potassium ion. CONCLUSIONS: This is the first crystallographic indication of the 'base-on' mode of cobalamin binding. An unusually long cobalt-base bond seems to favor homolytic cleavage of the cobalt-carbon bond and therefore to favor radical enzyme catalysis. Reactive radical intermediates can be protected from side reactions by spatial isolation inside the barrel. On the basis of unique direct interactions between the potassium ion and the two hydroxyl groups of the substrate, direct participation of a potassium ion in enzyme catalysis is strongly suggested.

Change of liver function in hypertrophying lobe of rabbit liver after portal branch ligation.

Preoperative portal embolization (PE) is useful for the prevention of postoperative liver failure after extended hepatectomy. However, clinical evaluation of liver function in the hypertrophying lobe after PE has not been studied. Here we report functional changes in the hypertrophying lobe using a 80% portal-branch-ligation rabbit model. Liver function was evaluated by the expression of liver-specific genes detected by Northern blot analysis and plasma disappearance rate of indocyanine green (ICG). The weight of the unligated lobe after portal ligation increased about twofold on the 7th postoperative day (POD) and about threefold on the 14th POD. The mRNA levels of the liver-specific genes (albumin, aldolase B, and tyrosine aminotransferase) in the unligated lobe decreased to about 50% on the 1st POD and returned to the preoperative levels on the 7-14th POD. In contrast, the expression of histone H2B mRNA increased on the 3rd-7th POD. The plasma disappearance rate of ICG (K-ICG) in the rabbit that has only the unligated lobe did not significantly change during the first 7 days, but then improved and recovered to 80% of that in the rabbit that has whole liver on the 14th POD. These results indicate that liver function of the hypertrophying lobe after portal branch ligation does not increase during the first 7 days despite an increase in liver weight. This finding suggests that the compensatory hypertrophying liver is enlarging without functional augmentation in the early period after PE.

Calcium signaling in cold cells studied in cultured dorsal root ganglion neurons.

Thermosensitive cold cells were identified in cultured dorsal root ganglion neurons from newborn rats. The neurons were loaded with a calcium indicator, Fura-PE3, and the change in intracellular Ca2+ concentration ([Ca2+]i) of the neurons was measured with microfluorimetry. Thirteen per cent of the cells responded to the cold stimulation. The diameter of the responder cells was 16.3+/-3.2 microm (mean+/-S.D., n = 25). The lowering of the temperature from 35 degrees C to 20 degrees C increased [Ca2+]i from 59.6+/-10.6 nM to 203.4+/-14.8 nM (n = 25). The [Ca2+]i response was dependent on the intensity of the cold stimulation. The depletion of extracellular Ca2+ diminished the Ca2+ elevation. However, a Na(+)-free condition did not influence the response. We concluded that the cold stimulation opens Ca2(+)-permeable channels in putative cold cells from dorsal root ganglion neurons.

Intracellular Mg2+ surge follows Ca2+ increase during depolarization in cultured neurons.

The intracellular magnesium and calcium concentrations in cultured dorsal root ganglion neurons were measured using a fluorescent Mg2+ indicator, Mag-Fura-2 and a Ca2+ indicator, Fura-2, respectively. The magnesium concentration in the cytoplasm was higher than that in the nuclei at rest; 0.68+/-0.10 mM (mean+/-S.E.M., n=7) in the cytoplasm and 0.11+/-0.05 mM in the nucleus. When depolarized by a 60 mM KCl solution, the magnesium concentration increased remarkably in the cytoplasm; 1.52+/-0.26 mM (n=7) in the cytoplasm and 0.25+/-0. 12 mM in the nucleus. This is in contrast to a Ca2+ increase due to depolarization in which the increase was remarkable also in the nucleus. The Mg2+ response displayed a rapid spontaneous recovery even in the presence of the high K+ solution. The Ca2+ response, on the other hand, accompanied a slow recovery 'plateau'. Simultaneous measurements of Mg2+ and Ca2+ by a double-labeling experiment revealed that the Ca2+ concentration started to rise 0.46+/-0.05 s (n=32) earlier, and it reached its peak 1.38+/-0.12 s (n=32) earlier than Mg2+. These results support the scheme of 'calcium induced magnesium release', that the depolarization-induced elevation of the Ca2+ concentration causes an increase in the Mg2+ concentration in the cytoplasm.

Crystallization and preliminary crystallographic studies of FMN-binding protein from Desulfovibrio vulgaris miyazaki F.

The flavin mononucleotide binding protein from Desulfovibrio vulgaris (Miyazaki F) was crystallized using the vapour-diffusion method. The crystal belongs to the monoclinic space group P21 with unit-cell parameters a = 37.2, b = 84.6, c = 41.1 A, beta = 94.1 degrees, contains two molecules per asymmetric unit and diffracts beyond 1.2 A resolution with a synchrotron radiation X-ray source.

Decreased expression of the peroxisomal bifunctional enzyme and carbonyl reductase in human hepatocellular carcinomas.

Human hepatocellular carcinomas (HCC) are known to frequently exhibit clear-cell or fatty change. The expression of three enzymes related to fatty acid metabolism, the peroxisomal bifunctional enzyme (enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, BE), cytosolic carbonyl reductase (CR) and the alpha-class glutathione S-transferase (GST) was investigated immunohistochemically in 45 HCC samples, to examine their relevance to this phenomenon and to antioxidant cellular defence. The tumour sizes ranged from 3 mm to 37 mm in diameter (mean 19 mm). Of 8 highly differentiated carcinomas (Edmondson's grade 1), 5 and 6 showed positive staining for BE and CR respectively, like the surrounding non-hepatoma tissues. Of 37 Edmondson's grade II-IV lesions, 31 exhibited negative or only weakly positive staining for both enzymes, as compared with the surrounding tissues. The combined rates for weakly positive and negative staining for BE or CR were proportional to the degree of dedifferentiation. However, 3 of 26 grade III tumours showed enhanced staining. Intensities of staining for CR were in accordance with those for BE in 40 of the total of 45 HCC. Immunoblot analysis also demonstrated concerted alteration of the two enzymes in carcinoma tissues. The staining of the alpha-class GST was hardly changed in Edmondson's grade I and II cases but was decreased in 24 of 31 grade III and IV lesions. The great majority of the BE-negative carcinomas did not demonstrate fatty or clear-cell change. These results suggested that BE and CR might be possible markers for the analysis of multistage hepatocarcinogenesis but that decrease or loss was not reflected in increased fat storage.

Crystallization and preliminary x-ray study of two crystal forms of Klebsiella oxytoca diol dehydratase-cyanocobalamin complex.

Two crystal forms of Klebsiella oxytoca diol dehydratase complexed with cyanocobalamin have been obtained and preliminary crystallographic experiments have been performed. The crystals belong to two different space groups, depending on the crystallization conditions. One crystal (form I) belongs to space group P212121 with unit-cell parameters a = 76.2, b = 122.3, c = 209. 6 A, and diffracts to 2.2 A resolution using an X-ray beam from a synchrotron radiation source. The other crystal (form II) belongs to space group P21 with unit-cell parameters a = 75.4, b = 132.7, c = 298.8 A, beta = 91.9 degrees, and diffracts to 3.0 A resolution. For the purpose of structure determination, a heavy-atom derivative search was carried out and some mercuric derivatives were found to be promising. Structure analysis by the multiple isomorphous replacement method is now under way.

Enhancement of the specific growth rate of Thiobacillus ferrooxidans by diatomaceous earth.

Diatomaceous earth is an effective carrier with a large surface area on which Thiobacillus ferrooxidans adsorbs, and enhances the oxidation rate of ferrous ions by the bacterium. The adsorption of T. ferrooxidans on diatomaceous earth was expressed by the Henry isotherm, and reached equilibrium within five minutes. The enhancement was due to an increase in the growth rate of the adsorbed cells, which were so active that their specific growth rate calculated using a Monod-type equation was higher than that of the free cells in the liquid phase.

Identification of L-Bornesitol and Changes in Its Content during Flower Bud Development in Sweet Pea (Lathyrus odoratus L.).

An unidentified carbohydrate was isolated from sweet pea (Lathyrus odoratus L. cv. Diana) petals using HPLC. The isolated compound was identified as L-1-O-methyl-myo-inositol, called L-bornesitol, using (1)H-NMR, (13)C-NMR, and CI-MS. L-Bornesitol was distributed in all organs at high concentrations. L-Bornesitol concentration of petals gradually decreased during flower bud development, but the L-bornesitol content increased by about 5 times.

Warm cells revealed by microfluorimetry of Ca2+ in cultured dorsal root ganglion neurons.

Warm cells were identified by Fura-PE3-based microfluorimetry of Ca2+ in cultured dorsal root ganglion (DRG) neurons. In response to a physiologically relevant stimulus temperature (43 degrees C), a subpopulation of small DRG neurons from new born rats increased the intracellular Ca2+ concentration ([Ca2+]i). Seven percent of the cells responded to the warm stimulus. The stimulus evoked elevation in [Ca2+]i from 52.5 +/- 9.5 nM (mean +/- S.D., n = 18) to 171.0 +/- 15.6 nM in cells between 15 and 25 microns in diameter. The depletion of extracellular Ca2+ diminished the Ca2+ elevation. The Na(+)-free condition also diminished the response. We concluded that the heat stimulation opens nonselective cation channels in putative warm cells from DRG neurons.

Depolarization triggers intracellular magnesium surge in cultured dorsal root ganglion neurons.

Intracellular magnesium concentration ([Mg2+]i) of cultured dorsal root ganglion (DRG) neurons was measured using the magnesium indicator Mag-Fura-2/AM. [Mg2+]i was 0.48 +/- 0.08 mM (mean +/- SEM, n = 23) at rest, and it increased 3-fold by depolarization with a 60-mM K+ solution. The [Mg2+]i increase was observed in the absence of extracellular Mg2+, but the increase disappeared in the absence of extracellular Ca2+. 50 microM cadmium or 100 microM verapamil, a Ca2+ channel blocker, also diminished the rise of [Mg2+]i. The additional measurement of an intracellular Ca2+ concentration ([Ca2+]i) indicated that the [Mg2+]i rise requires a threshold concentration of [Ca2+]i to be reached; above 60 nM. The present results indicate that depolarization induces a Ca2+-influx through voltage dependent Ca channels and this causes the release of Mg2+ from intracellular stores into the cytoplasm.